Novel DNA cloning method

ABSTRACT

The invention refers to a novel method for cloning DNA molecules using a homologous recombination mechanism between at least two DNA molecules comprising: a) providing a host cell capable of performing homologous recombination, b) contacting in said host cell a first DNA molecule which is capable of being replicated in said host cell with a second DNA molecule comprising at least two regions of sequence homology to regions on the first DNA molecule, under conditions which favour homologous recombination between said first and second DNA molecules and c) selecting a host cell in which homologous recombination between said first and second DNA molecules has occurred.

DESCRIPTION

[0001] The invention refers to a novel method for cloning DNA molecules using a homologous recombination mechanism between at least two DNA molecules. Further, novel reagent kits suitable for DNA cloning are provided.

[0002] Current methods for cloning foreign DNA in bacterial cells usually comprise the steps of providing a suitable bacterial vector, cleaving said vector with a restriction enzyme and in vitro-inserting a foreign DNA fragment in said vector. The resulting recombinant vectors are then used to transform bacteria. Although such cloning methods have been used successfully for about 20 years they suffer from several drawbacks. These drawbacks are, in particular, that the in vitro steps required for inserting foreign DNA in a vector are often very complicated and time-consuming, if no suitable restriction sites are available on the foreign DNA or the vector.

[0003] Furthermore, current methods usually rely on the presence of suitable restriction enzyme cleavage sites in the vector into which the foreign DNA fragment is placed. This imposes two limitations on the final cloning product. First, the foreign DNA fragment can usually only be inserted into the vector at the position of such a restriction site or sites. Thus, the cloning product is limited by the disposition of suitable restriction sites and cloning into regions of the vector where there is no suitable restriction site, is difficult and often imprecise. Second, since restriction sites are typically 4 to 8 base pairs in length, they occur a multiple number of times as the size of the DNA molecules being used increases. This represents a practical limitation to the size of the DNA molecules that can be manipulated by most current cloning techniques. In particular, the larger sizes of DNA cloned into vectors such as cosmids, BACs, PACs and P1s are such that it is usually impractical to manipulate them directly by restriction enzyme based techniques. Therefore, there is a need for providing a new cloning method, from which the drawbacks of the prior art have at least partly been eliminated.

[0004] According to the present invention it was found that an efficient homologous recombination mechanism between two DNA molecules occurs at usable frequencies in a bacterial host cell which is capable of expressing the products of the recE and recT genes or functionally related genes such as the redα and redβ genes, or the phage P22 recombination system (Kolodner et al., Mol.Microbiol. 11 (1994) 23-30; Fenton, A. C. and Poteete, A. R., Virology 134 (1984) 148-160; Poteete, A. R. and Fenton, A. C., Virology 134 (1984) 161-167). This novel method of cloning DNA fragments is termed “ET cloning”.

[0005] The identification and characterization of the E. coli RecE and RecT proteins is described Gillen et al. (J.Bacteriol. 145 (1981), 521-532) and Hall et al. (J.Bacteriol. 175 (1993), 277-287). Hall and Kolodner (Proc.Natl.Acad.Sci. USA 91 (1994), 3205-3209) disclose in vitro homologous pairing and strand exchange of linear double-stranded DNA and homologous circular single-stranded DNA promoted by the RecT protein. Any references to the use of this method for the cloning of DNA molecules in cells cannot be found therein.

[0006] The recET pathway of genetic recombination in E. coli is known (Hall and Kolodner (1994), supra; Gillen et al. (1981), supra). This pathway requires the expression of two genes, recE and recT. The DNA sequence of these genes has been published (Hall et al., supra). The RecE protein is similar to bacteriophage proteins, such as λ exo or λ Redα (Gillen et al., J.Mol.Biol. 113 (1977), 27-41; Little, J.Biol.Chem. 242 (1967), 679-686; Radding and Carter, J.Biol.Chem. 246 (1971), 2513-2518; Joseph and Kolodner, J.Biol.Chem. 258 (1983), 10418-10424). The RecT protein is similar to bacteriophage proteins, such as λ β-protein or λ Redβ (Hall et al. (1993), supra; Muniyappa and Radding, J.Biol.Chem. 261 (1986), 7472-7478; Kmiec and Hollomon, J.Biol.Chem. 256 (1981), 12636-12639). The content of the above-cited documents is incorporated herein by reference.

[0007] Oliner et al. (Nucl.Acids Res. 21 (1993), 5192-5197) describe in vivo cloning of PCR products in E. coli by intermolecular homologous recombination between a linear PCR product and a linearized plasmid vector. Other previous attempts to develop new cloning methods based on homologous recombination in prokaryotes, too, relied on the use of restriction enzymes to linearise the vector (Bubeck et al., Nucleic Acids Res. 21 (1 993), 3601-3602; Oliner et al., Nucleic Acids Res. 21 (1993), 5192-5197; Degryse, Gene 170 (1996), 45-50) or on the host-specific recA-dependent recombination system (Hamilton et al., J.Bacteriol. 171 (1989), 4617-4622; Yang et al., Nature Biotech. 15 (1997), 859-865; Dabert and Smith, Genetics 145 (1997), 877-889). These methods are of very limited applicability and are hardly used in practice.

[0008] The novel method of cloning DNA according to the present invention does not require in vitro treatments with restriction enzymes or DNA ligases and is therefore fundamentally distinct from the standard methodologies of DNA cloning. The method relies on a pathway of homologous recombination in E. coli involving the recE and recT gene products, or the redα and redβ gene products, or functionally equivalent gene products. The method covalently combines one preferably linear and preferably extrachromosomal DNA fragment, the DNA fragment to be cloned, with one second preferably circular DNA vector molecule, either an episome or the endogenous host chromosome or chromosomes. It is therefore distinct from previous descriptions of cloning in E. coli by homologous recombination which either rely on the use of two linear DNA fragments or different recombination pathways.

[0009] The present invention provides a flexible way to use homologous recombination to engineer large DNA molecules including an intact>76 kb plasmid and the E. coli chromosome. Thus, there is practically no limitation of target choice either according to size or site. Therefore, any recipient DNA in a host cell, from high copy plasmid to the genome, is amenable to precise alteration. In addition to engineering large DNA molecules, the invention outlines new, restriction enzyme-independent approaches to DNA design. For example, deletions between any two chosen base pairs in a target episome can be made by choice of oligonucleotide homology arms. Similarly, chosen DNA sequences can be inserted at a chosen base pair to create, for example, altered protein reading frames. Concerted combinations of insertions and deletions, as well as point mutations, are also possible. The application of these strategies is particularly relevant to complex or difficult DNA constructions, for example, those intended for homologous recombinations in eukaryotic cells, e.g. mouse embryonic stem cells. Further, the present invention provides a simple way to position site specific recombination target sites exactly where desired. This will simplify applications of site specific recombination in other living systems, such as plants and mice.

[0010] A subject matter of the present invention is a method for cloning DNA molecules in cells comprising the steps:

[0011] a) providing a host cell capable of performing homologous recombination,

[0012] b) contacting in said host cell a first DNA molecule which is capable of being replicated in said host cell with a second DNA molecule comprising at least two regions of sequence homology to regions on the first DNA molecule, under conditions which favour homologous recombination between said first and second DNA molecules and

[0013] c) selecting a host cell in which homologous recombination between said first and second DNA molecules has occurred.

[0014] In the method of the present invention the homologous recombination preferably occurs via the recET mechanism, i.e. the homologous recombination is mediated by the gene products of the recE and the recT genes which are preferably selected from the E. coli genes recE and recT or functionally related genes such as the phage λ redα and redβ genes.

[0015] The host cell suitable for the method of the present invention preferably is a bacterial cell, e.g. a gram-negative bacterial cell. More preferably, the host cell is an enterobacterial cell, such as Salmonella, Klebsiella or Escherichia. Most preferably the host cell is an Escherichia coli cell. It should be noted, however, that the cloning method of the present invention is also suitable for eukaryotic cells, such as fungi, plant or animal cells.

[0016] Preferably, the host cell used for homologous recombination and propagation of the cloned DNA can be any cell, e.g. a bacterial strain in which the products of the recE and recT, or redα and redβ, genes are expressed. The host cell may comprise the recE and recT genes located on the host cell chromosome or on non-chromosomal DNA, preferably on a vector, e.g. a plasmid. In a preferred case, the RecE and RecT, or Redα and Redβ, gene products are expressed from two different regulatable promoters, such as the arabinose-inducible BAD promoter or the lac promoter or from non-regulatable promoters. Alternatively, the recE and recT, or redα and redβ, genes are expressed on a polycistronic mRNA from a single regulatable or non-regulatable promoter. Preferably the expression is controlled by regulatable promoters.

[0017] Especially preferred is also an embodiment, wherein the recE or redα gene is expressed by a regulatable promoter. Thus, the recombinogenic potential of the system is only elicited when required and, at other times, possible undesired recombination reactions are limited. The recT or redβ gene, on the other hand, is preferably overexpressed with respect to recE or redo. This may be accomplished by using a strong constitutive promoter, e.g. the EM7 promoter and/or by using a higher copy number of recT, or redβ, versus recE, or redα, genes.

[0018] For the purpose of the present invention any recE and recT genes are suitable insofar as they allow a homologous recombination of first and second DNA molecules with sufficient efficiency to give rise to recombination products in more than 1 in 10⁹ cells transfected with DNA. The recE and recT genes may be derived from any bacterial strain or from bacteriophages or may be mutants and variants thereof. Preferred are recE and recT genes which are derived from E. coli or from E. coli bacteriophages, such as the redα and redβ genes from lambdoid phages, e.g. bacteriophage λ.

[0019] More preferably, the recE or redα gene is selected from a nucleic acid molecule comprising

[0020] (a) the nucleic acid sequence from position 1320 (ATG) to 2159 (GAC) as depicted in FIG. 7B,

[0021] (b) the nucleic acid sequence from position 1320 (ATG) to 1 998(CGA) as depicted in FIG. 14B,

[0022] (c) a nucleic acid encoding the same polypeptide within the degeneracy of the genetic code and/or

[0023] (d) a nucleic acid sequence which hybridizes under stringent conditions with the nucleic acid sequence from (a), (b) and/or (c).

[0024] More preferably, the recT or redβ gene is selected from a nucleic acid molecule comprising

[0025] (a) the nucleic acid sequence from position 2155 (ATG) to 2961 (GAA) as depicted in FIG. 7B,

[0026] (b) the nucleic acid sequence from position 2086 (ATG) to 2868 (GCA) as depicted in FIG. 14B,

[0027] (c) a nucleic acid encoding the same polypeptide within the degeneracy of the genetic code and/or

[0028] (d) a nucleic acid sequence which hybridizes under stringent conditions with the nucleic acid sequences from (a), (b) and/or (c).

[0029] It should be noted that the present invention also encompasses mutants and variants of the given sequences, e.g. naturally occurring mutants and variants or mutants and variants obtained by genetic engineering. Further it should be noted that the recE gene depicted in FIG. 7B is an already truncated gene encoding amino acids 588-866 of the native protein. Mutants and variants preferably have a nucleotide sequence identity of at least 60%, preferably of at least 70% and more preferably of at least 80% of the recE and recT sequences depicted in FIGS. 7B and 13B, and of the redα and redβ sequences depicted in FIG. 14B.

[0030] According to the present invention hybridization under stringent conditions preferably is defined according to Sambrook et al. (1989), infra, and comprises a detectable hybridization signal after washing for 30 min in 0.1×SSC, 0.5% SDS at 55° C., preferably at 62° C. and more preferably at 68° C.

[0031] In a preferred case the recE and recT genes are derived from the corresponding endogenous genes present in the E. coli K12 strain and its derivatives or from bacteriophages. In particular, strains that carry the sbcA mutation are suitable. Examples of such strains are JC8679 and JC 9604 (Gillen et al. (1981), supra). Alternatively, the corresponding genes may also be obtained from other coliphages such as lambdoid phages or phage P22.

[0032] The genotype of JC 8679 and JC 9604 is Sex (Hfr, F+, F−, or F′): F−.JC 8679 comprises the mutations: recBC 21, recC 22, sbcA 23, thr-1, ara-1 4, leu B 6, DE (gpt-proA) 62, lacY1, tsx-33, gluV44 (AS), galK2 (Oc), LAM-, his-60, relA 1, rps L31 (strR), xyl A5, mtl-1, argE3 (Oc) and thi-1 . JC 9604 comprises the same mutations and further the mutation recA 56.

[0033] Further, it should be noted that the recE and recT, or redα and redβ, genes can be isolated from a first donor source, e.g. a donor bacterial cell and transformed into a second receptor source, e.g. a receptor bacterial or eukaryotic cell in which they are expressed by recombinant DNA means.

[0034] In one embodiment of the invention, the host cell used is a bacterial strain having an sbcA mutation, e.g. one of E. coli strains JC 8679 and JC 9604 mentioned above. However, the method of the invention is not limited to host cells having an sbcA mutation or analogous cells. Surprisingly, it has been found that the cloning method of the invention also works in cells without sbcA mutation, whether recBC+ or recBC−, e.g. also in prokaryotic recBC+ host cells, e.g. in E. coli recBC+ cells. In that case preferably those host cells are used in which the product of a recBC type exonuclease inhibitor gene is expressed. Preferably, the exonuclease inhibitor is capable of inhibiting the host recBC system or an equivalent thereof. A suitable example of such exonuclease inhibitor gene is the λ redγ gene (Murphy, J.Bacteriol. 173 (1991), 5808-5821) and functional equivalents thereof, respectively, which, for example, can be obtained from other coliphages such as from phage P22 (Murphy, J.Biol.Chem. 269 (1994), 22507-22516).

[0035] More preferably, the exonuclease inhibitor gene is selected from a nucleic acid molecule comprising

[0036] (a) the nucleic acid sequence from position 3588 (ATG) to 4002 (GTA) as depicted in FIG. 14A,

[0037] (b) a nucleic acid encoding the same polypeptide within the degeneracy of the genetic code and/or

[0038] (c) a nucleic acid sequence which hybridizes under stringent conditions (as defined above) with the nucleic acid sequence from (a) and/ or (b).

[0039] Surprisingly, it has been found that the expression of an exonuclease inhibitor gene in both recBC+ and recBC− strains leads to significant improvement of cloning efficiency.

[0040] The cloning method according to the present invention employs a homologous recombination between a first DNA molecule and a second DNA molecule. The first DNA molecule can be any DNA molecule that carries an origin of replication which is operative in the host cell, e.g. an E. coli replication origin. Further, the first DNA molecule is present in a form which is capable of being replicated in the host cell. The first DNA molecule, i.e. the vector, can be any extrachromosomal DNA molecule containing an origin of replication which is operative in said host cell, e.g. a plasmid including single, low, medium or high copy plasmids or other extrachromosomal circular DNA molecules based on cosmid, P1, BAC or PAC vector technology. Examples of such vectors are described, for example, by Sambrook et al. (Molecular Cloning, Laboratory Manual, 2nd Edition (1989), Cold Spring Harbor Laboratory Press) and loannou et al. (Nature Genet. 6 (1994), 84-89) or references cited therein. The first DNA molecule can also be a host cell chromosome, particularly the E. coli chromosome. Preferably, the first DNA molecule is a double-stranded DNA molecule.

[0041] The second DNA molecule is preferably a linear DNA molecule and comprises at least two regions of sequence homology, preferably of sequence identity to regions on the first DNA molecule. These homology or identity regions are preferably at least 15 nucleotides each, more preferably at least 20 nucleotides and, most preferably, at least 30 nucleotides each.

[0042] Especially good results were obtained when using sequence homology regions having a length of about 40 or more nucleotides, e.g. 60 or more nucleotides. The two sequence homology regions can be located on the linear DNA fragment so that one is at one end and the other is at the other end, however they may also be located internally. Preferably, also the second DNA molecule is a double-stranded DNA molecule.

[0043] The two sequence homology regions are chosen according to the experimental design. There are no limitations on which regions of the first DNA molecule can be chosen for the two sequence homology regions located on the second DNA molecule, except that the homologous recombination event cannot delete the origin of replication of the first DNA molecule. The sequence homology regions can be interrupted by non-identical sequence regions as long as sufficient sequence homology is retained for the homologous recombination reaction. By using sequence homology arms having non-identical sequence regions compared to the target site mutations such as substitutions, e.g. point mutations, insertions and/or deletions may be introduced into the target site by ET cloning.

[0044] The second foreign DNA molecule which is to be cloned in the bacterial cell may be derived from any source. For example, the second DNA molecule may be synthesized by a nucleic acid amplification reaction such as a PCR where both of the DNA oligonucleotides used to prime the amplification contain in addition to sequences at the 3′-ends that serve as a primer for the amplification, one or the other of the two homology regions. Using oligonucleotides of this design, the DNA product of the amplification can be any DNA sequence suitable for amplification and will additionally have a sequence homology region at each end.

[0045] A specific example of the generation of the second DNA molecule is the amplification of a gene that serves to convey a phenotypic difference to the bacterial host cells, in particular, antibiotic resistance. A simple variation of this procedure involves the use of oligonucleotides that include other sequences in addition to the PCR primer sequence and the sequence homology region. A further simple variation is the use of more than two amplification primers to generate the amplification product. A further simple variation is the use of more than one amplification reaction to generate the amplification product. A further variation is the use of DNA fragments obtained by methods other than PCR, for example, by endonuclease or restriction enzyme cleavage to linearize fragments from any source of DNA.

[0046] It should be noted that the second DNA molecule is not necessarily a single species of DNA molecule. It is of course possible to use a heterogenous population of second DNA molecules, e.g. to generate a DNA library, such as a genomic or cDNA library.

[0047] The method of the present invention may comprise the contacting of the first and second DNA molecules in vivo. In one embodiment of the present invention the second DNA fragment is transformed into a bacterial strain that already harbors the first vector DNA molecule. In a different embodiment, the second DNA molecule and the first DNA molecule are mixed together in vitro before co-transformation in the bacterial host cell. These two embodiments of the present invention are schematically depicted in FIG. 1. The method of transformation can be any method known in the art (e.g. Sambrook et al. supra). The preferred method of transformation or co-transformation, however, is electroporation.

[0048] After contacting the first and second DNA molecules under conditions which favour homologous recombination between first and second DNA molecules via the ET cloning mechanism a host cell is selected, in which homologous recombination between said first and second DNA molecules has occurred. This selection procedure can be carried out by several different methods. In the following three preferred selection methods are depicted in FIG. 2 and described in detail below.

[0049] In a first selection method a second DNA fragment is employed which carries a gene for a marker placed between the two regions of sequence homology wherein homologous recombination is detectable by expression of the marker gene. The marker gene may be a gene for a phenotypic marker which is not expressed in the host or from the first DNA molecule. Upon recombination by ET cloning, the change in phenotype of the host strain conveyed by the stable acquisition of the second DNA fragment identifies the ET cloning product.

[0050] In a preferred case, the phenotypic marker is a gene that conveys resistance to an antibiotic, in particular, genes that convey resistance to kanamycin, ampillicin, chloramphenicol, tetracyclin or any other substance that shows bacteriocidal or bacteriostatic effects on the bacterial strain employed.

[0051] A simple variation is the use of a gene that complements a deficiency present within the bacterial host strain employed. For example, the host strain may be mutated so that it is incapable of growth without a metabolic supplement. In the absence of this supplement, a gene on the second DNA fragment can complement the mutational defect thus permitting growth. Only those cells which contain the episome carrying the intended DNA rearrangement caused by the ET cloning step will grow.

[0052] In another example, the host strain carries a phenotypic marker gene which is mutated so that one of its codons is a stop codon that truncates the open reading frame. Expression of the full length protein from this phenotypic marker gene requires the introduction of a suppressor tRNA gene which, once expressed, recognizes the stop codon and permits translation of the full open reading frame. The suppressor tRNA gene is introduced by the ET cloning step and successful recombinants identified by selection for, or identification of, the expression of the phenotypic marker gene. In these cases, only those cells which contain the intended DNA rearrangement caused by the ET cloning step will grow.

[0053] A further simple variation is the use of a reporter gene that conveys a readily detectable change in colony colour or morphology. In a preferred case, the green fluorescence protein (GFP) can be used and colonies carrying the ET cloning product identified by the fluorescence emissions of GFP. In another preferred case, the lacZ gene can be used and colonies carrying the ET cloning product identified by a blue colony colour when X-gal is added to the culture medium.

[0054] In a second selection method the insertion of the second DNA fragment into the first DNA molecule by ET cloning alters the expression of a marker present on the first DNA molecule. In this embodiment the first DNA molecule contains at least one marker gene between the two regions of sequence homology and homologous recombination may be detected by an altered expression, e.g. lack of expression of the marker gene.

[0055] In a preferred application, the marker present on the first DNA molecule is a counter-selectable gene product, such as the sacB, ccdB or tetracycline-resistance genes. In these cases, bacterial cells that carry the first DNA molecule unmodified by the ET cloning step after transformation with the second DNA fragment, or co-transformation with the second DNA fragment and the first DNA molecule, are plated onto a medium so the expression of the counter-selectable marker conveys a toxic or bacteriostatic effect on the host. Only those bacterial cells which contain the first DNA molecule carrying the intended DNA rearrangement caused by the ET cloning step will grow.

[0056] In another preferred application, the first DNA molecule carries a reporter gene that conveys a readily detectable change in colony colour or morphology. In a preferred case, the green fluorescence protein (GFP) can be present on the first DNA molecule and colonies carrying the first DNA molecule with or without the ET cloning product can be distinguished by differences in the fluorescence emissions of GFP. In another preferred case, the lacZ gene can be present on the first DNA molecule and colonies carrying the first DNA molecule with or without the ET cloning product identified by a blue or white colony colour when X-gal is added to the culture medium.

[0057] In a third selection method the integration of the second DNA fragment into the first DNA molecule by ET cloning removes a target site for a site specific recombinase, termed here an RT (for recombinase target) present on the first DNA molecule between the two regions of sequence homology. A homologous recombination event may be detected by removal of the target site.

[0058] In the absence of the ET cloning product, the RT is available for use by the corresponding site specific recombinase. The difference between the presence or not of this RT is the basis for selection of the ET cloning product. In the presence of this RT and the corresponding site specific recombinase, the site specific recombinase mediates recombination at this RT and changes the phenotype of the host so that it is either not able to grow or presents a readily observable phenotype. In the absence of this RT, the corresponding site specific recombinase is not able to mediate recombination.

[0059] In a preferred case, the first DNA molecule to which the second DNA fragment is directed, contains two RTs, one of which is adjacent to, but not part of, an antibiotic resistance gene. The second DNA fragment is directed, by design, to remove this RT. Upon exposure to the corresponding site specific recombinase, those first DNA molecules that do not carry the ET cloning product will be subject to a site specific recombination reaction between the RTs that remove the antibiotic resistance gene and therefore the first DNA molecule fails to convey resistance to the corresponding antibiotic. Only those first DNA molecules that contain the ET cloning product, or have failed to be site specifically recombined for some other reason, will convey resistance to the antibiotic.

[0060] In another preferred case, the RT to be removed by ET cloning of the second DNA fragment is adjacent to a gene that complements a deficiency present within the host strain employed. In another preferred case, the RT to be removed by ET cloning of the second DNA fragment is adjacent to a reporter gene that conveys a readily detectable change in colony colour or morphology.

[0061] In another preferred case, the RT to be removed by ET cloning of the second DNA fragment is anywhere on a first episomal DNA molecule and the episome carries an origin of replication incompatible with survival of the bacterial host cell if it is integrated into the host genome. In this case the host genome carries a second RT, which may or may not be a mutated RT so that the corresponding site specific recombinase can integrate the episome, via its RT, into the RT sited in the host genome. Other preferred RTs include RTs for site specific recombinases of the resolvase/transposase class. RTs include those described from existing examples of site specific recombination as well as natural or mutated variations thereof.

[0062] The preferred site specific recombinases include Cre, FLP, Kw or any site specific recombinase of the integrase class. Other preferred site specific recombinases include site specific recombinases of the resolvase/transposase class.

[0063] There are no limitations on the method of expression of the site specific recombinase in the host cell. In a preferred method, the expression of the site specific recombinase is regulated so that expression can be induced and quenched according to the optimisation of the ET cloning efficiency. In this case, the site specific recombinase gene can be either integrated into the host genome or carried on an episome. In another preferred case, the site specific recombinase is expressed from an episome that carries a conditional origin of replication so that it can be eliminated from the host cell.

[0064] In another preferred case, at least two of the above three selection methods are combined. A particularly preferred case involves a two-step use of the first selection method above, followed by use of the second selection method. This combined use requires, most simply, that the DNA fragment to be cloned includes a gene, or genes that permits the identification, in the first step, of correct ET cloning products by the acquisition of a phenotypic change. In a second step, expression of the gene or genes introduced in the first step is altered so that a second round of ET cloning products can be identified. In a preferred example, the gene employed is the tetracycline resistance gene and the first step ET cloning products are identified by the acquisition of tetracycline resistance. In the second step, loss of expression of the tetracycline gene is identified by loss of sensitivity to nickel chloride, fusaric acid or any other agent that is toxic to the host cell when the tetracycline gene is expressed. This two-step procedure permits the identification of ET cloning products by first the integration of a gene that conveys a phenotypic change on the host, and second by the loss of a related phenotypic change, most simply by removal of some of the DNA sequences integrated in the first step. Thereby the genes used to identify ET cloning products can be inserted and then removed to leave ET cloning products that are free of these genes.

[0065] In a further embodiment of the present invention the ET cloning may also be used for a recombination method comprising the steps of

[0066] a) providing a source of RecE and RecT, or Redα and Redβ, proteins,

[0067] b) contacting a first DNA molecule which is capable of being replicated in a suitable host cell with a second DNA molecule comprising at least two regions of sequence homology to regions on the first DNA molecule, under conditions which favour homologous recombination between said first and second DNA molecules and

[0068] c) selecting DNA molecules in which a homologous recombination between said first and second DNA molecules has occurred.

[0069] The source of RecE and RecT, or Redα and Redβ, proteins may be either purified or partially purified RecE and RecT, or Redα and Redβ, proteins or cell extracts comprising RecE and RecT, or Redα and Redβ, proteins.

[0070] The homologous recombination event in this embodiment may occur in vitro, e.g. when providing a cell extract containing further components required for homologous recombination. The homologous recombination event, however, may also occur in vivo, e.g. by introducing RecE and RecT, or Redα and Redβ, proteins or the extract in a host cell (which may be recET positive or not, or redαβ positive or not) and contacting the DNA molecules in the host cell. When the recombination occurs in vitro the selection of DNA molecules may be accomplished by transforming the recombination mixture in a suitable host cell and selecting for positive clones as described above. When the recombination occurs in vivo the selection methods as described above may directly be applied.

[0071] A further subject matter of the invention is the use of cells, preferably bacterial cells, most preferably, E. coli cells capable of expressing the recE and recT, or redα and redβ, genes as a host cell for a cloning method involving homologous recombination.

[0072] Still a further subject matter of the invention is a vector system capable of expressing recE and recT, or redα and redβ, genes in a host cell and its use for a cloning method involving homologous recombination. Preferably, the vector system is also capable of expressing an exonuclease inhibitor gene as defined above, e.g. the λ redγ gene. The vector system may comprise at least one vector. The recE and recT, or redα and redβ, genes are preferably located on a single vector and more preferably under control of a regulatable promoter which may be the same for both genes or a single promoter for each gene. Especially preferred is a vector system which is capable of overexpressing the recT, or redβ, gene versus the recE, or redα, gene.

[0073] Still a further subject matter of the invention is the use of a source of RecE and RecT, or Redα and Redβ, proteins for a cloning method involving homologous recombination.

[0074] A still further subject matter of the invention is a reagent kit for cloning comprising

[0075] (a) a host cell, preferably a bacterial host cell,

[0076] (b) means of expressing recE and recT, or redα and redβ, genes in said host cell, e.g. comprising a vector system, and

[0077] (c) a recipient cloning vehicle, e.g. a vector, capable of being replicated in said cell.

[0078] On the one hand, the recipient cloning vehicle which corresponds to the first DNA molecule of the process of the invention can already be present in the bacterial cell. On the other hand, it can be present separated from the bacterial cell.

[0079] In a further embodiment the reagent kit comprises

[0080] (a) a source for RecE and RecT, or Redα and Redβ, proteins and

[0081] (b) a recipient cloning vehicle capable of being propagated in a host cell and

[0082] (c) optionally a host cell suitable for propagating said recipient cloning vehicle.

[0083] The reagent kit furthermore contains, preferably, means for expressing a site specific recombinase in said host cell, in particular, when the recipient ET cloning product contains at least one site specific recombinase target site. Moreover, the reagent kit can also contain DNA molecules suitable for use as a source of linear DNA fragments used for ET cloning, preferably by serving as templates for PCR generation of the linear fragment, also as specifically designed DNA vectors from which the linear DNA fragment is released by restriction enzyme cleavage, or as prepared linear fragments included in the kit for use as positive controls or other tasks. Moreover, the reagent kit can also contain nucleic acid amplification primers comprising a region of homology to said vector. Preferably, this region of homology is located at the 5′-end of the nucleic acid amplification primer.

[0084] The invention is further illustrated by the following Sequence listings, Figures and Examples.

[0085] SEQ ID NO. 1: shows the nucleic acid sequence of the plasmid pBAD24-rec ET (FIG. 7).

[0086] SEQ ID NOs 2/3: show the nucleic acid and amino acid sequences of the truncated recE gene (t-recE) present on pBAD24-recET at positions 1320-2162.

[0087] SEQ ID NOs 4/5: show the nucleic acid and amino acid sequences of the recT gene present on pBAD24-recET at position 21 55-2972.

[0088] SEQ ID NOs 6/7: show the nucleic acid and amino acid sequences of the araC gene present on the complementary stand to the one shown of pBAD24-recET at positions 974-996.

[0089] SEQ ID NOs 8/9: show the nucleic acid an amino acid sequences of the bla gene present on pBAD24-recET at positions 3493-4353.

[0090] SEQ ID NO 10: shows the nucleic acid sequence of the plasmid pBAD-ETy (FIG. 13).

[0091] SEQ ID No 11: shows the nucleic acid sequence of the plasmid pBAD-αβγ (FIG. 14) as well as the coding regions for the genes redα (1320-200), redβ (2086-2871) and redγ (3403-3819).

[0092] SEQ ID NOs 12-14: show the amino acid sequences of the Redα, Redβ and Redγ proteins, respectively. The redγ sequence is present on each of pBAD-ETγ (FIG. 13) and pBAD-αβγ (FIG. 14).

[0093]FIG. 1

[0094] A preferred method for ET cloning is shown by diagram. The linear DNA fragment to be cloned is synthesized by PCR using oligonucleotide primers that contain a left homology arm chosen to match sequences in the recipient episome and a sequence for priming in the PCR reaction, and a right homology arm chosen to match another sequence in the recipient episome and a sequence for priming in the PCR reaction. The product of the PCR reaction, here a selectable marker gene (sm1), is consequently flanked by the left and right homology arms and can be mixed together in vitro with the episome before co-transformation, or transformed into a host cell harboring the target episome. The host cell contains the products of the recE and recT genes. ET cloning products are identified by the combination of two selectable markers, sm1 and sm2 on the recipient episome.

[0095]FIG. 2

[0096] Three ways to identify ET cloning products are depicted. The first, (on the left of the figure), shows the acquisition, by ET cloning, of a gene that conveys a phenotypic difference to the host, here a selectable marker gene (sm). The second (in the centre of the figure) shows the loss, by ET cloning, of a gene that conveys a phenotypic difference to the host, here a counter selectable marker gene (counter-sm). The third shows the loss of a target site (RT, shown as triangles on the circular episome) for a site specific recombinase (SSR), by ET cloning. In this case, the correct ET cloning product deletes one of the target sites required by the SSR to delete a selectable marker gene (sm). The failure of the SSR to delete the sm gene identifies the correct ET cloning product.

[0097]FIG. 3

[0098] A simple example of ET cloning is presented.

[0099] (a) Top panel—PCR products (left lane) synthesized from oligonucleotides designed as described in FIG. 1 to amplify by PCR a kanamycin resistance gene and to be flanked by homology arms present in the recipient vector, were mixed in vitro with the recipient vector (2nd lane) and cotransformed into a recET+ E. coli host. The recipient vector carried an ampillicin resistance gene. (b) Transformation of the sbcA E. coli strain JC9604 with either the PCR product alone (0.2 μg) or the vector alone (0.3 μg) did not convey resistance to double selection with ampicillin and kanamycin (amp+kan), however cotransformation of both the PCR product and the vector produced double resistant colonies. More than 95% of these colonies contained the correct ET cloning product where the kanamycin gene had precisely integrated into the recipient vector according to the choice of homology arms. The two lanes on the right of (a) show Pvu II restriction enzyme digestion of the recipient vector before and after ET cloning. (c) As for b, except that six PCR products (0.2 μg each) were cotransformed with pSVpaZ11 (0.3 μg each) into JC9604 and plated onto Amp+Kan plates or Amp plates. Results are plotted as Amp+ Kan-resistant colonies, representing recombination products, divided by Amp-resistant colonies, representing the plasmid transformation efficiency of the competent cell preparation, ×10⁶. The PCR products were equivalent to the a-b PCR product except that homology arm lengths were varied. Results are from five experiments that used the same batches of competent cells and DNAs. Error bars represent standard deviation. (d) Eight products flanked by 50 bp homology arms were cotransformed with pSVpaZ11 into JC9604. All eight PCR products contained the same left homology arm and amplified neo gene. The right homology arms were chosen from the pSVpaZ11 sequence to be adjacent to (0), or at increasing distances (7-3100 bp), from the left. Results are from four experiments.

[0100]FIG. 4

[0101] ET cloning in an approximately 100 kb P1 vector to exchange the selectable marker.

[0102] A P1 clone which uses a kanamycin resistance gene as selectable marker and which contains at least 70 kb of the mouse Hox a gene cluster was used. Before ET cloning, this episome conveys kanamycin resistance (top panel, upper left) to its host E. coli which are ampillicin sensitive (top panel, upper right). A linear DNA fragment designed to replace the kanamycin resistance gene with an ampillicin resistance gene was made by PCR as outlined in FIG. 1 and transformed into E. coli host cells in which the recipient Hox a/P1 vector was resident. ET cloning resulted in the deletion of the kanamycin resistance gene, and restoration of kanamycin sensitivity (top panel, lower left) and the acquisition of ampillicin resistance (top panel, lower right). Precise DNA recombination was verified by restriction digestion and Southern blotting analyses of isolated DNA before and after ET cloning (lower panel).

[0103]FIG. 5

[0104] ET cloning to remove a counter selectable marker

[0105] A PCR fragment (upper panel, left, third lane) made as outlined in FIGS. 1 and 2 to contain the kanamycin resistance gene was directed by its chosen homology arms to delete the counter selectable ccdB gene present in the vector, pZero-2.1. The PCR product and the pZero vector were mixed in vitro (upper panel, left, 1 st lane) before cotransformation into a recE/recT+ E. coli host. Transformation of pZero-2.1 alone and plating onto kanamycin selection medium resulted in little colony growth (lower panel, left). Cotransformation of pZero-2.1 and the PCR product presented ET cloning products (lower panel, right) which showed the intended molecular event as visualized by Pvu II digestion (upper panel, right).

[0106]FIG. 6

[0107] ET cloning mediated by inducible expression of recE and recT from an episome.

[0108] RecE/RecT mediate homologous recombination between linear and circular DNA molecules. (a) The plasmid pBAD24-recET was transformed into E. coli JC5547, and then batches of competent cells were prepared after induction of RecE/RecT expression by addition of L-arabinose for the times indicated before harvesting. A PCR product, made using oligonucleotides e and f to contain the chloramphenicol resistance gene (cm) of pMAK705 and 50 bp homology arms chosen to flank the ampicillin resistance gene (bla) of pBAD24-recET, was then transformed and recombinants identified on chloramphenicol plates. (b) Arabinose was added to cultures of pBAD24-recET transformed JC5547 for different times immediately before harvesting for competent cell preparation. Total protein expression was analyzed by SDS-PAGE and Coomassie blue staining. (c) The number of chloramphenicol resistant colonies per μg of PCR product was normalized against a control for transformation efficiency, determined by including 5 pg pZero2.1, conveying kanamycin resistance, in the transformation and plating an aliquot onto Kan plates.

[0109]FIG. 7A

[0110] The plasmid pBAD24-recET is shown by diagram. The plasmid contains the genes recE (in a truncated form) and recT under control of the inducible BAD promoter (P_(BAD)) The plasmid further contains an ampillicin resistance gene (Amp^(r)) and an araC gene.

[0111]FIG. 7B

[0112] The nucleic acid sequence and the protein coding portions of pBAD24-recET are depicted.

[0113]FIG. 8

[0114] Manipulation of a large E. coli episome by multiple recombination steps. a Scheme of the recombination reactions. A P1 clone of the Mouse Hoxa complex, resident in JC9604, was modified by recombination with PCR products that contained the neo gene and two Flp recombination targets (FRTs). The two PCR products were identical except that one was flanked by g and h homology arms (insertion), and the other was flanked by i and h homology arms (deletion). In a second step, the neo gene was removed by Flp recombination between the FRTs by transient transformation of a FIp expression plasmid based on the pSC101 temperature-sensitive origin (ts ori). b Upper panel; ethidium bromide stained agarose gel showing EcoR1 digestions of P1 DNA preparations from three independent colonies for each step. Middle panel; a Southern blot of the upper panel hybridized with a neo gene probe. Lower panel; a Southern blot of the upper panel hybridized with a Hoxa3 probe to visualize the site of recombination. Lanes 1, the original Hoxa3 P1 clone grown in E. coli strain NS3145. Lanes 2, replacement of the Tn903 kanamycin resistance gene resident in the P1 vector with an ampicillin resistance gene increased the 8.1 kb band (lanes 1), to 9.0 kb. Lanes 3, insertion of the Tn5-neo gene with g-h homology arms upstream of Hoxa3, increased the 6.7 kb band (lanes 1, 2) to 9.0 kb. Lanes 4, Flp recombinase deleted the g-h neo gene reducing the 9.0 kb band (lanes 3) back to 6.7 kb. Lanes 5, deletion of 6 kb of Hoxa3-4 intergenic DNA by replacement with the i-h neo gene, decreased the 6.7 kb band (lanes 2) to 4.5 kb. Lanes 6, Flp recombinase deleted the i-h neo gene reducing the 4.5 kb band to 2.3 kb.

[0115]FIG. 9

[0116] Manipulation of the E. coli chromosome. A Scheme of the recombination reactions. The endogenous lacZ gene of JC9604 at 7.8′ of the E. coli chromosome, shown in expanded form with relevant Ava I sites and coordinates, was targeted by a PCR fragment that contained the neo gene flanked by homology arms j and k, and loxP sites, as depicted. Integration of the neo gene removed most of the lacZ gene including an Ava I site to alter the 1443 and 3027 bp bands into a 3277 bp band. In a second step, the neo gene was removed by Cre recombination between the loxPs by transient transformation of a Cre expression plasmid based on the PSC101 temperature-sensitive origin (ts ori). Removal of the neo gene by Cre recombinase reduces the 3277 band to 2111 bp. b β-galactosidase expression evaluated by streaking colonies on X-Gal plates. The top row of three streaks show β-galactosidase expression in the host JC9604 strain (w.t.), the lower three rows (Km) show 24 independent primary colonies, 20 of which display a loss of β-galactosidase expression indicactive of the intended recombination event. c Southern analysis of E. coli chromosomal DNA digested with Ava I using a random primed probe made from the entire lacZ coding region; lanes 1, 2, w.t.; lanes 3-6, four independent white colonies after integration of the j-k neo gene; lanes 7-10; the same four colonies after transient transformation with the Cre expression plasmid.

[0117]FIG. 10

[0118] Two rounds of ET cloning to introduce a point mutation. a Scheme of the recombination reactions. The lacZ gene of pSVpaX1 was disrupted in JC9604lacZ, a strain made by the experiment of FIG. 9 to ablate endogenous lacZ expression and remove competitive sequences, by a sacB-neo gene cassette, synthesized by PCR to plB279 and flanked by l and m homology arms. The recombinants, termed pSV-sacB-neo, were selected on Amp+Kan plates. The lacZ gene of pSV-sacB-neo was then repaired by a PCR fragment made from the intact lacZ gene using l® and m® homology arms. The m® homology arm included a silent C to G change that created a BamH1 site. The recombinants, termed pSVpaX1®, were identified by counter selection against the sacB gene using 7% sucrose. b β-galactosidase expression from pSVpaX1 was disrupted in pSV-sacB-neo and restored in pSVpaX1®. Expression was analyzed on X-gal plates. Three independent colonies of each pSV-sacB-neo and pSVpaX1® are shown. c Ethidium bromide stained agarose gels of BamH1 digested DNA prepared from independent colonies taken after counter selection with sucrose. All β-galactosidase expressing colonies (blue) contained the introduced BamH 1 restriction site (upper panel). All white colonies displayed large rearrangements and no product carried the diagnostic 1.5kb BamH1 restriction fragment (lower panel).

[0119]FIG. 11

[0120] Transferance of ET cloning into a recBC+ host to modify a large episome. a Scheme of the plasmid, pBAD-ETγ, which carries the mobile ET system, and the strategy employed to target the Hoxa P1 episome. pBAD-ETγ is based on pBAD24 and includes (i) the truncated recE gene (t-recE) under the arabinose-inducible P_(BAD) promoter; (ii) the recT gene under the EM7 promoter; and (iii) the redγ gene under the Tn5 promoter. It was transformed into NS31 45, a recA E. coli strain which contained the Hoxa P1 episome. After arabinose induction, competent cells were prepared and transformed with a PCR product carrying the chloramphenicol resistance gene (cm) flanked by n and p homology arms. n and p were chosen to recombine with a segment of the P1 vector. b Southern blots of Pvu II digested DNAs hybridized with a probe made from the P1 vector to visualize the recombination target site (upper panel) and a probe made from the chloramphenicol resistance gene (lower panel). Lane 1, DNA prepared from cells harboring the Hoxa P1 episome before ET cloning. Lanes 2-17, DNA prepared from 16 independent chloramphenicol resistant colonies.

[0121]FIG. 12

[0122] Comparison of ET cloning using the recE/recT genes in pBAD-ETγ with redα/redβ genes in pBAD-αβγ.

[0123] The plasmids pBAD-ETγ or pBAD-αβγ, depicted, were transformed into the E. coli recA−, recBC+ strain, DK1 and targeted by a chloramphenicol gene as described in FIG. 6 to evaluate ET cloning efficiencies. Arabinose induction of protein expression was for 1 hour.

[0124]FIG. 13A

[0125] The plasmid pBAD-ETγ is shown by diagram.

[0126]FIG. 13B

[0127] The nucleic acid sequence and the protein coding portions of pBAD-ETγ are depicted.

[0128]FIG. 14A

[0129] The plasmid pBAD-αβγ is shown by diagram. This plasmid substantially corresponds to the plasmid shown in FIG. 13 except that the recE and recT genes are substituted by the redα and redβ genes.

[0130]FIG. 14B

[0131] The nucleic acid sequence and the protein coding portions of pBAD-αβγ are depicted.

[0132] 1. Methods

[0133] 1.1. Preparation of Linear Fragments

[0134] Standard PCR reaction conditions were used to amplify linear DNA fragments. The sequences of the primers used are depicted in Table 1.

[0135] Table 1

[0136] The Tn5-neo gene from pJP5603 (Penfold and Pemberton, Gene 118 (1992), 145-146) was amplified by using oligo pairs a/b and c/d. The chloramphenicol (cm) resistant gene from pMAK705 (Hashimoto-Gotoh and Sekiguchi, J.Bacteriol. 131 (1977), 405-412) was amplified by using primer pairs e/f and n/p. The Tn5-neo gene flanked by FRT or loxP sites was amplified from pKaZ or pKaX (http://www.embl-heidelberg.de/Externailnfo/stewart) using oligo pairs i/h, g/h and j/k. The sacB-neo cassette from plB279 (Blomfield et al., Mol.Microbiol. 5 (1991), 1447-1457) was amplified by using oligo pair l/m. The lacZ gene fragment from pSVpaZ11 (Buchholz et al., Nucleic Acids Res. 24 (1996), 4256-4262) was amplified using oligo pair l® /m®. PCR products were purified using the QIAGEN PCR Purification Kit and eluted with H₂O₂, followed by digestion of any residual template DNA with Dpn 1. After digestion, PCR products were extracted once with Phenol:CHCl₃, ethanol precipitated and resuspended in H₂O at approximately 0.5 μg/μl.

[0137] 1.2 Preparation of Competent Cells and Electroporation

[0138] Saturated overnight cultures were diluted 50 fold into LB medium, grown to an OD600 of 0.5, following by chilling on ice for 15 min. Bacterial cells were centrifuged at 7,000 rpm for 10 min at 0° C. The pellet was resuspended in ice-cold 10% glycerol and centrifuged again (7,000 rpm, −5° C., 10 min). This was repeated twice more and the cell pellet was suspended in an equal volume of ice-cold 10% glycerol. Aliquots of 50 μl were frozen in liquid nitrogen and stored at −80° C. Cells were thawed on ice and 1 μl DNA solution (containing, for co-transformation, 0.3 μg plasmid and 0.2 μg PCR products; or, for transformation, 0.2μg PCR products) was added. Electroporation was performed using ice-cold cuvettes and a Bio-Rad Gene Pulser set to 25 μFD, 2.3 kV with Pulse Controller set at 200 ohms. LB medium (1 ml) was added after electroporation. The cells were incubated at 37° C. for 1 hour with shaking and then spread on antibiotic plates.

[0139] 1.3 Induction of RecE and RecT Expression

[0140]E. coli JC5547 carrying pBAD24-recET was cultured overnight in LB medium plus 0.2% glucose, 100 μg/ml ampicillin. Five parallel LB cultures, one of which (0) included 0.2% glucose, were started by a 1/100 inoculation. The cultures were incubated at 37° C. with shaking for 4 hours and 0.1% L-arabinose was added 3, 2, 1 or ½ hour before harvesting and processing as above. Immediately before harvesting, 100 μl was removed for analysis on a 10% SDS-polyacrylamide gel. E. coli NS3145 carrying Hoxa-P1 and pBAD-ETγ was induced by 0.1% L-arabinose for 90 min before harvesting.

[0141] 1.4 Transient Transformation of FLP and Cre Expression Plasmids

[0142] The FLP and Cre expression plasmids, 705-Cre and 705-FLP (Buchholz et al., Nucleic Acids Res. 24 (1996), 3118-3119), based on the pSC101 temperature sensitive origin, were transformed into rubidium chloride competent bacterial cells. Cells were spread on 25 μg/ml chloramphenicol plates, and grown for 2 days at 30° C., whereupon colonies were picked, replated on L-agar plates without any antibiotics and incubated at 40° C. overnight. Single colonies were analyzed on various antibiotic plates and all showed the expected loss of chloramphenicol and kanamycin resistance.

[0143] 1.5 Sucrose Counter Selection of sacB Expression

[0144] The E. coli JC9604lacZ strain, generated as described in FIG. 11, was cotransformed with a sacB-neo PCR fragment and pSVpaX1 (Buchholz et al, Nucleic Acids Res. 24 (1996), 4256-4262). After selection on 100 μg/ml ampicillin, 50 μg/ml kanamycin plates, pSVpaX-sacB-neo plasmids were isolated and cotransformed into fresh JC9604lacZ cells with a PCR fragment amplified from pSVpaX1 using primers l^(i·)/m^(i·). Oligo m^(i·) carried a silent point mutation which generated a BamHI site. Cells were plated on 7% sucrose, 100 μg/ml ampicillin, 40 μg/ml X-gal plates and incubated at 28° C. for 2 days. The blue and white colonies grown on sucrose plates were counted and further checked by restriction analysis.

[0145] 1.6 Other Methods

[0146] DNA preparation and Southern analysis were performed according to standard procedures. Hybridization probes were generated by random priming of fragments isolated from the Tn5 neo gene (Pvull), Hoxa3 gene (both Hindill fragments), lacZ genes (EcoR1 and BamH1 fragments from pSVpaX1), cm gene (BstB1 fragments from pMAK705) and P1 vector fragments (2.2 kb EcoR1 fragments from P1 vector).

[0147] 2. Results

[0148] 2.1 Identification of Recombination Events in E. coli

[0149] To identify a flexible homologous recombination reaction in E. coli, an assay based on recombination between linear and circular DNAs was designed (FIG. 1, FIG. 3). Linear DNA carrying the Tn5 kanamycin resistance gene (neo) was made by PCR (FIG. 3a). Initially, the oligonucleotides used for PCR amplification of neo were 60 mers consisting of 42 nucleotides at their 5′ ends identical to chosen regions in the plasmid and, at the 3′ ends, 18 nucleotides to serve as PCR primers. Linear and circular DNAs were mixed in equimolar proportions and co-transformed into a variety of E. coli hosts. Homologous recombination was only detected in sbcA E. coli hosts. More than 95% of double ampicillin/kanamycin resistant colonies (FIG. 3b) contained the expected homologously recombined plasmid as determined by restriction digestion and sequencing. Only a low background of kanamycin resistance, due to genomic integration of the neo gene, was apparent (not shown).

[0150] The linear plus circular recombination reaction was characterized in two ways. The relationship between homology arm length and recombination efficiency was simple, with longer arms recombining more efficiently (FIG. 3c). Efficiency increased within the range tested, up to 60 bp. The effect of distance between the two chosen homology sites in the recipient plasmid was examined (FIG. 3d). A set of eight PCR fragments was generated by use of a constant left homology arm with differing right homology arms. The right homology arms were chosen from the plasmid sequence to be 0-3100 bp from the left. Correct products were readily obtained from all, with less than 4 fold difference between them, although the insertional product (0) was least efficient. Correct products also depended on the presence of both homology arms, since PCR fragments containing only one arm failed to work.

[0151] 2.2 Involvement of RecE and RecT

[0152] The relationship between hostgenotype and this homologous recombination reaction was more systematically examined using a panel of E. coli strains deficient in various recombination components (Table 2).

[0153] Table 2

[0154] Only the two sbcA strains, JC8679 and JC9604 presented the intended recombination products and RecA was not required. In sbcA strains, expression of RecE and RecT is activated. Dependence on recE can be inferred from comparison of JC8679 with JC8691. Notably no recombination products were observed in JC9387 suggesting that the sbcBC background is not capable of supporting homologous recombination based on 50 nucleotide homology arms.

[0155] To demonstrate that RecE and RecT are involved, part of the recET operon was cloned into an inducible expression vector to create pBAD24-recET (FIG. 6a). the recE gene was truncated at its N-terminal end, as the first 588 a.a.s of RecE are dispensable. The recBC strain, JC5547, was transformed with pBAD24-recET and a time course of RecE/RecT induction performed by adding arabinose to the culture media at various times before harvesting for competent cells. The batches of harvested competent cells were evaluated for protein expression by gel electrophoresis (FIG. 6b) and for recombination between a linear DNA fragment and the endogenous pBAD24-recET plasmid (FIG. 6c). Without induction of RecE/RecT, no recombinant products were found, whereas recombination increased in approximate concordance with increased RecE/RecT expression. This experiment also shows that co-transformation of linear and circular DNAs is not essential and the circular recipient can be endogenous in the host. From the results shown in FIGS. 3, 6 and Table 2, we conclude that RecE and RecT mediate a very useful homologous recombination reaction in recBC E. coli at workable frequencies. Since RecE and RecTare involved, we refer to this way of recombining linear and circular DNA fragments as “ET cloning ”.

[0156] 2.3 Application of ET Cloning to Large Target DNAs

[0157] To show that large DNA episomes could be manipulated in E. coli, a>76 kb P1 clone that contains at least 59 kb of the intact mouse Hoxa complex, (confirmed bv DNA sequencing and Southern blotting), was transferred to an E. coli strain having an sbcA background (JC9604) and subjected to two rounds of ET cloning. In the first round, the Tn903 kanamycin resistance gene resident in the P1 vector was replaced by an ampicillin resistance gene (FIG. 4). In the second round, the interval between the Hoxa3 and a4 genes was targeted either by inserting the neo gene between two base pairs upstream of the Hoxa3 proximal promoter, or by deleting 6203 bp between the Hoxa3 and a4 genes (FIG. 8a). Both insertional and deletional ET cloning products were readily obtained (FIG. 8b, lanes 2, 3 and 5) showing that the two rounds of ET cloning took place in this large E. coli episome with precision and no apparent unintended recombination.

[0158] The general applicability of ET cloning was further examined by targeting a gene in the E. coli chromosome (FIG. 9a). The β-galactosidase (lacZ) gene of JC9604 was chosen so that the ratio between correct and incorrect recombinants could be determined by evaluating β-galactosidase expression. Standard conditions (0.2 μg PCR fragment; 50 μl competent cells), produced 24 primary colonies, 20 of which were correct as determined by β-galactosidase expression (FIG. 9b), and DNA analysis (FIG. 9c, lanes 3-6).

[0159] 2.4 Secondary Recombination Reactions to Remove Operational Sequences

[0160] The products of ET cloning as described above are limited by the necessary inclusion of selectable marker genes. Two different ways to use a further recombination step to remove this limitation were developed. In the first way, site specific recombination mediated by either Flp or Cre recombinase was employed. In the experiments of FIGS. 8 and 9, either Flp recombination target sites (FRTs) or Cre recombination target sites (loxPs) were included to flank the neo gene in the linear substrates. Recombination between the FRTs or loxPs was accomplished by Flp or Cre, respectively, expressed from plasmids with the pSC101 temperature sensitive replication origin (Hashimoto-Gotoh and Sekiguchi, J.Bacteriol. 131 (1977), 405-412) to permit simple elimination of these plasmids after site specific recombination by temperature shift. The precisely recombined Hoxa P1 vector was recovered after both ET and Flp recombination with no other recombination products apparent (FIG. 8, lanes 4 and 6). Similarly, Cre recombinase precisely recombined the targeted lacZ allele (FIG. 9, lanes 7-10). Thus site specific recombination can be readily coupled with ET cloning to remove operational sequences and leave a 34 bp site specific recombination target site at the point of DNA manipulation.

[0161] In the second way to remove the selectable marker gene, two rounds of ET cloning, combining positive and counter selection steps, were used to leave the DNA product free of any operational sequences (FIG. 10a).

[0162] Additionally this experiment was designed to evaluate, by a functional test based on β-galactosidase activity, whether ET cloning promoted small mutations such as frame shift or point mutations within the region being manipulated. In the first round, the lacZ gene of pSVpaXl was disrupted with a 3.3 kb PCR fragment carrying the neo and B. subtilis sacB (Blomfield et al., Mol.Microbiol. 5 (1991), 1447-1457) genes, by selection for kanamycin resistance (FIG. 10a). As shown above for other positively selected recombination products, virtually all selected colonies were white (FIG. 10b), indicative of successful lacZ disruption, and 17 of 17 were confirmed as correct recombinants by DNA analysis. In the second round, a 1.5 kb PCR fragment designed to repair lacZ was introduced by counter selection against the sacB gene. Repair of lacZ included a silent point mutation to create a BamH1 restriction site. Approximately one quarter of sucrose resistant colonies expressed β-galactosidase, and all analyzed (17 of 17; FIG. 10c) carried the repaired lacZ gene with the BamH1 point mutation. The remaining three quarters of sucrose resistant colonies did not express β-galactosidase, and all analyzed (17 of 17; FIG. 10c) had undergone a variety of large mutational events, none of which resembled the ET cloning product. Thus, in two rounds of ET cloning directed at the lacZ gene, no disturbances of β-galactosidase activity by small mutations were observed, indicating the RecE/RecT recombination works with high fidelity. The significant presence of incorrect products observed in the counter selection step is an inherent limitation of the use of counter selection, since any mutation that ablates expression of the counter selection gene will be selected. Notably, all incorrect products were large mutations and therefore easily distinguished from the correct ET product by DNA analysis. In a different experiment (FIG. 5), we observed that ET cloning into pZero2.1 (InVitroGen) by counter selection against the ccdB gene gave a lower background of incorrect products (8%), indicating that the counter selection background is variable according to parameters that differ from those that influence ET cloning efficiencies.

[0163] 2.5 Transference of ET Cloning Between E. coli Hosts

[0164] The experiments shown above were performed in recBC-E. coli hosts since the sbcA mutation had been identified as a suppressor of recBC (Barbour et al., Proc.Natl.Acad.Sci. USA 67 (1970), 128-135; Clark, Genetics 78 (1974), 259-271). However, many useful E. coli strains are recBC+, including strains commonly used for propagation of P1, BAC or PAC episomes. To transfer ET cloning into recBC+ strains, we developed pBAD-ETγ and pBAD-αβγ (FIGS. 13 and 14). These plasmids incorporate three features important to the mobility of ET cloning. First, RecBC is the major E. coli exonuclease and degrades introduced linear fragments. Therefore the RecBC inhibitor, Redy (Murphy, J.Bacteriol. 173 (1991), 5808-5821), was included. Second, the recombinogenic potential of RecE/RecT, or Redα/Redβ, was regulated by placing recE or redα under an inducible promoter. Consequently ET cloning can be induced when required and undesired recombination events which are restricted at other times. Third, we observed that ET cloning efficiencies are enhanced when RecT, or Redβ, but not RecE, or Redα, is overexpressed. Therefore we placed recT, or redβ, under the strong, constitutive, EM7 promoter.

[0165] pBAD-ETγ was transformed into NS3145 E. coli harboring the original Hoxa P1 episome (FIG. 11a). A region in the P1 vector backbone was targeted by PCR amplification of the chloramphenicol resistance gene (cm) flanked by n and p homology arms. As described above for positively selected ET cloning reactions, most (>90%) chloramphenicol resistant colonies were correct. Notably, the overall efficiency of ET cloning, in terms of linear DNA transformed, was nearly three times better using pBAD-ETγ than with similar experiments based on targeting the same episome in the sbcA host, JC9604. This is consistent with our observation that overexpression of RecT improves ET cloning efficiencies.

[0166] A comparison between ET cloning efficiencies mediated by RecE/RecT, expressed from pBAD-ETγ, and Redα/Redβ, expressed from pBAD-αβγ was made in the recA−, recBC+ E. coli strain, DK1 (FIG. 12). After transformation of E. coli DK1 with either pBAD-ETγ or pBAD-αβγ, the same experiment as described in FIGS. 6a,c, to replace the bla gene of the pBAD vector with a chloramphenicol gene was performed. Both pBAD-ETγ or pBAD-αβγ presented similar ET cloning efficiencies in terms of responsiveness to arabinose induction of RecE and Redα, and number of targeted events. TABLE 2 E. coli Amp Strains Genotypes Amp + Kan ×10⁸/μg JC8679 recBC sbcA 318 2.30 JC9604 recA recBC sbcA 114 0.30 JC8691 recBC sbcA recE 0 0.37 JC5547 recA recBC 0 0.37 JC5519 recBC 0 1.80 JC15329 recA recBC sbcBC 0 0.03 JC9387 recBC sbcBC 0 2.20 JC8111 recBC sbcBC recF 0 2.40 JC9366 recA 0 0.37 JC13031 recJ 0 0.45

[0167]

1 14 1 6150 DNA Artificial Sequence misc_feature (1)..(6150) plasmid pBAD24-rec ET 1 atcgatgcat aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac 60 tccgtcaagc cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca 120 ttcacttttt cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta 180 aatacccgcg agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata 240 ggcatccggg tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag 300 cttaagacgc taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag 360 caaacatgct gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg 420 tactgacaag cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct 480 tccatgcgcc gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc 540 ccttcccctt gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc 600 gcttcatccg ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca 660 tgccagtagg cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga 720 tgacgaccgt agtgatgaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa 780 acaaattctc gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata 840 taacctttca ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc 900 ggcgttaaac ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt 960 tgcgcttcag ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat 1020 tgcatcagac attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta 1080 accccgctta ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt 1140 aacaaaagtg tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca 1200 ctttgctatg ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta 1260 tcgcaactct ctactgtttc tccatacccg tttttttggg ctagcaggag gaattcacca 1320 tggatcccgt aatcgtagaa gacatagagc caggtattta ttacggaatt tcgaatgaga 1380 attaccacgc gggtcccggt atcagtaagt ctcagctcga tgacattgct gatactccgg 1440 cactatattt gtggcgtaaa aatgcccccg tggacaccac aaagacaaaa acgctcgatt 1500 taggaactgc tttccactgc cgggtacttg aaccggaaga attcagtaac cgctttatcg 1560 tagcacctga atttaaccgc cgtacaaacg ccggaaaaga agaagagaaa gcgtttctga 1620 tggaatgcgc aagcacagga aaaacggtta tcactgcgga agaaggccgg aaaattgaac 1680 tcatgtatca aagcgttatg gctttgccgc tggggcaatg gcttgttgaa agcgccggac 1740 acgctgaatc atcaatttac tgggaagatc ctgaaacagg aattttgtgt cggtgccgtc 1800 cggacaaaat tatccctgaa tttcactgga tcatggacgt gaaaactacg gcggatattc 1860 aacgattcaa aaccgcttat tacgactacc gctatcacgt tcaggatgca ttctacagtg 1920 acggttatga agcacagttt ggagtgcagc caactttcgt ttttctggtt gccagcacaa 1980 ctattgaatg cggacgttat ccggttgaaa ttttcatgat gggcgaagaa gcaaaactgg 2040 caggtcaaca ggaatatcac cgcaatctgc gaaccctgtc tgactgcctg aataccgatg 2100 aatggccagc tattaagaca ttatcactgc cccgctgggc taaggaatat gcaaatgact 2160 aagcaaccac caatcgcaaa agccgatctg caaaaaactc agggaaaccg tgcaccagca 2220 gcagttaaaa atagcgacgt gattagtttt attaaccagc catcaatgaa agagcaactg 2280 gcagcagctc ttccacgcca tatgacggct gaacgtatga tccgtatcgc caccacagaa 2340 attcgtaaag ttccggcgtt aggaaactgt gacactatga gttttgtcag tgcgatcgta 2400 cagtgttcac agctcggact tgagccaggt agcgccctcg gtcatgcata tttactgcct 2460 tttggtaata aaaacgaaaa gagcggtaaa aagaacgttc agctaatcat tggctatcgc 2520 ggcatgattg atctggctcg ccgttctggt caaatcgcca gcctgtcagc ccgtgttgtc 2580 cgtgaaggtg acgagtttag cttcgaattt ggccttgatg aaaagttaat acaccgcccg 2640 ggagaaaacg aagatgcccc ggttacccac gtctatgctg tcgcaagact gaaagacgga 2700 ggtactcagt ttgaagttat gacgcgcaaa cagattgagc tggtgcgcag cctgagtaaa 2760 gctggtaata acgggccgtg ggtaactcac tgggaagaaa tggcaaagaa aacggctatt 2820 cgtcgcctgt tcaaatattt gcccgtatca attgagatcc agcgtgcagt atcaatggat 2880 gaaaaggaac cactgacaat cgatcctgca gattcctctg tattaaccgg ggaatacagt 2940 gtaatcgata attcagagga atagatctaa gcttggctgt tttggcggat gagagaagat 3000 tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc 3060 tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg 3120 tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa 3180 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga 3240 acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc 3300 ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg 3360 ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt tttctaaata 3420 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 3480 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 3540 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 3600 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 3660 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 3720 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct 3780 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 3840 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 3900 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 3960 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 4020 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta 4080 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 4140 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 4200 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 4260 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 4320 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata 4380 ctttagattg atttacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 4440 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc 4500 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt 4560 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg 4620 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac 4680 gttctttaat agtggactct tgttccaaac ttgaacaaca ctcaacccta tctcgggcta 4740 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat 4800 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt aaaaggatct 4860 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 4920 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 4980 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 5040 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 5100 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 5160 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 5220 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 5280 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 5340 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 5400 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 5460 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 5520 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 5580 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg 5640 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc 5700 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc 5760 atctgtgcgg tatttcacac cgcatagggt catggctgcg ccccgacacc cgccaacacc 5820 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac 5880 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca 5940 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga 6000 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga 6060 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt 6120 agaggatctg ctcatgtttg acagcttatc 6150 2 843 DNA Artificial Sequence misc_feature (1)..(843) t-recE on plasmid pBAD24-recET at 1320-2162 2 atg gat ccc gta atc gta gaa gac ata gag cca ggt att tat tac gga 48 Met Asp Pro Val Ile Val Glu Asp Ile Glu Pro Gly Ile Tyr Tyr Gly 1 5 10 15 att tcg aat gag aat tac cac gcg ggt ccc ggt atc agt aag tct cag 96 Ile Ser Asn Glu Asn Tyr His Ala Gly Pro Gly Ile Ser Lys Ser Gln 20 25 30 ctc gat gac att gct gat act ccg gca cta tat ttg tgg cgt aaa aat 144 Leu Asp Asp Ile Ala Asp Thr Pro Ala Leu Tyr Leu Trp Arg Lys Asn 35 40 45 gcc ccc gtg gac acc aca aag aca aaa acg ctc gat tta gga act gct 192 Ala Pro Val Asp Thr Thr Lys Thr Lys Thr Leu Asp Leu Gly Thr Ala 50 55 60 ttc cac tgc cgg gta ctt gaa ccg gaa gaa ttc agt aac cgc ttt atc 240 Phe His Cys Arg Val Leu Glu Pro Glu Glu Phe Ser Asn Arg Phe Ile 65 70 75 80 gta gca cct gaa ttt aac cgc cgt aca aac gcc gga aaa gaa gaa gag 288 Val Ala Pro Glu Phe Asn Arg Arg Thr Asn Ala Gly Lys Glu Glu Glu 85 90 95 aaa gcg ttt ctg atg gaa tgc gca agc aca gga aaa acg gtt atc act 336 Lys Ala Phe Leu Met Glu Cys Ala Ser Thr Gly Lys Thr Val Ile Thr 100 105 110 gcg gaa gaa ggc cgg aaa att gaa ctc atg tat caa agc gtt atg gct 384 Ala Glu Glu Gly Arg Lys Ile Glu Leu Met Tyr Gln Ser Val Met Ala 115 120 125 ttg ccg ctg ggg caa tgg ctt gtt gaa agc gcc gga cac gct gaa tca 432 Leu Pro Leu Gly Gln Trp Leu Val Glu Ser Ala Gly His Ala Glu Ser 130 135 140 tca att tac tgg gaa gat cct gaa aca gga att ttg tgt cgg tgc cgt 480 Ser Ile Tyr Trp Glu Asp Pro Glu Thr Gly Ile Leu Cys Arg Cys Arg 145 150 155 160 ccg gac aaa att atc cct gaa ttt cac tgg atc atg gac gtg aaa act 528 Pro Asp Lys Ile Ile Pro Glu Phe His Trp Ile Met Asp Val Lys Thr 165 170 175 acg gcg gat att caa cga ttc aaa acc gct tat tac gac tac cgc tat 576 Thr Ala Asp Ile Gln Arg Phe Lys Thr Ala Tyr Tyr Asp Tyr Arg Tyr 180 185 190 cac gtt cag gat gca ttc tac agt gac ggt tat gaa gca cag ttt gga 624 His Val Gln Asp Ala Phe Tyr Ser Asp Gly Tyr Glu Ala Gln Phe Gly 195 200 205 gtg cag cca act ttc gtt ttt ctg gtt gcc agc aca act att gaa tgc 672 Val Gln Pro Thr Phe Val Phe Leu Val Ala Ser Thr Thr Ile Glu Cys 210 215 220 gga cgt tat ccg gtt gaa att ttc atg atg ggc gaa gaa gca aaa ctg 720 Gly Arg Tyr Pro Val Glu Ile Phe Met Met Gly Glu Glu Ala Lys Leu 225 230 235 240 gca ggt caa cag gaa tat cac cgc aat ctg cga acc ctg tct gac tgc 768 Ala Gly Gln Gln Glu Tyr His Arg Asn Leu Arg Thr Leu Ser Asp Cys 245 250 255 ctg aat acc gat gaa tgg cca gct att aag aca tta tca ctg ccc cgc 816 Leu Asn Thr Asp Glu Trp Pro Ala Ile Lys Thr Leu Ser Leu Pro Arg 260 265 270 tgg gct aag gaa tat gca aat gac taa 843 Trp Ala Lys Glu Tyr Ala Asn Asp * 275 280 3 280 PRT Artificial Sequence misc_feature (1)..(280) t-recE on plasmid pBAD24-recET at 1320-2162 3 Met Asp Pro Val Ile Val Glu Asp Ile Glu Pro Gly Ile Tyr Tyr Gly 1 5 10 15 Ile Ser Asn Glu Asn Tyr His Ala Gly Pro Gly Ile Ser Lys Ser Gln 20 25 30 Leu Asp Asp Ile Ala Asp Thr Pro Ala Leu Tyr Leu Trp Arg Lys Asn 35 40 45 Ala Pro Val Asp Thr Thr Lys Thr Lys Thr Leu Asp Leu Gly Thr Ala 50 55 60 Phe His Cys Arg Val Leu Glu Pro Glu Glu Phe Ser Asn Arg Phe Ile 65 70 75 80 Val Ala Pro Glu Phe Asn Arg Arg Thr Asn Ala Gly Lys Glu Glu Glu 85 90 95 Lys Ala Phe Leu Met Glu Cys Ala Ser Thr Gly Lys Thr Val Ile Thr 100 105 110 Ala Glu Glu Gly Arg Lys Ile Glu Leu Met Tyr Gln Ser Val Met Ala 115 120 125 Leu Pro Leu Gly Gln Trp Leu Val Glu Ser Ala Gly His Ala Glu Ser 130 135 140 Ser Ile Tyr Trp Glu Asp Pro Glu Thr Gly Ile Leu Cys Arg Cys Arg 145 150 155 160 Pro Asp Lys Ile Ile Pro Glu Phe His Trp Ile Met Asp Val Lys Thr 165 170 175 Thr Ala Asp Ile Gln Arg Phe Lys Thr Ala Tyr Tyr Asp Tyr Arg Tyr 180 185 190 His Val Gln Asp Ala Phe Tyr Ser Asp Gly Tyr Glu Ala Gln Phe Gly 195 200 205 Val Gln Pro Thr Phe Val Phe Leu Val Ala Ser Thr Thr Ile Glu Cys 210 215 220 Gly Arg Tyr Pro Val Glu Ile Phe Met Met Gly Glu Glu Ala Lys Leu 225 230 235 240 Ala Gly Gln Gln Glu Tyr His Arg Asn Leu Arg Thr Leu Ser Asp Cys 245 250 255 Leu Asn Thr Asp Glu Trp Pro Ala Ile Lys Thr Leu Ser Leu Pro Arg 260 265 270 Trp Ala Lys Glu Tyr Ala Asn Asp 275 280 4 810 DNA Artificial Sequence misc_feature (1)..(810) recT on plasmid pBAD24-recET at 2155-2972 4 atg act aag caa cca cca atc gca aaa gcc gat ctg caa aaa act cag 48 Met Thr Lys Gln Pro Pro Ile Ala Lys Ala Asp Leu Gln Lys Thr Gln 285 290 295 gga aac cgt gca cca gca gca gtt aaa aat agc gac gtg att agt ttt 96 Gly Asn Arg Ala Pro Ala Ala Val Lys Asn Ser Asp Val Ile Ser Phe 300 305 310 att aac cag cca tca atg aaa gag caa ctg gca gca gct ctt cca cgc 144 Ile Asn Gln Pro Ser Met Lys Glu Gln Leu Ala Ala Ala Leu Pro Arg 315 320 325 cat atg acg gct gaa cgt atg atc cgt atc gcc acc aca gaa att cgt 192 His Met Thr Ala Glu Arg Met Ile Arg Ile Ala Thr Thr Glu Ile Arg 330 335 340 345 aaa gtt ccg gcg tta gga aac tgt gac act atg agt ttt gtc agt gcg 240 Lys Val Pro Ala Leu Gly Asn Cys Asp Thr Met Ser Phe Val Ser Ala 350 355 360 atc gta cag tgt tca cag ctc gga ctt gag cca ggt agc gcc ctc ggt 288 Ile Val Gln Cys Ser Gln Leu Gly Leu Glu Pro Gly Ser Ala Leu Gly 365 370 375 cat gca tat tta ctg cct ttt ggt aat aaa aac gaa aag agc ggt aaa 336 His Ala Tyr Leu Leu Pro Phe Gly Asn Lys Asn Glu Lys Ser Gly Lys 380 385 390 aag aac gtt cag cta atc att ggc tat cgc ggc atg att gat ctg gct 384 Lys Asn Val Gln Leu Ile Ile Gly Tyr Arg Gly Met Ile Asp Leu Ala 395 400 405 cgc cgt tct ggt caa atc gcc agc ctg tca gcc cgt gtt gtc cgt gaa 432 Arg Arg Ser Gly Gln Ile Ala Ser Leu Ser Ala Arg Val Val Arg Glu 410 415 420 425 ggt gac gag ttt agc ttc gaa ttt ggc ctt gat gaa aag tta ata cac 480 Gly Asp Glu Phe Ser Phe Glu Phe Gly Leu Asp Glu Lys Leu Ile His 430 435 440 cgc ccg gga gaa aac gaa gat gcc ccg gtt acc cac gtc tat gct gtc 528 Arg Pro Gly Glu Asn Glu Asp Ala Pro Val Thr His Val Tyr Ala Val 445 450 455 gca aga ctg aaa gac gga ggt act cag ttt gaa gtt atg acg cgc aaa 576 Ala Arg Leu Lys Asp Gly Gly Thr Gln Phe Glu Val Met Thr Arg Lys 460 465 470 cag att gag ctg gtg cgc agc ctg agt aaa gct ggt aat aac ggg ccg 624 Gln Ile Glu Leu Val Arg Ser Leu Ser Lys Ala Gly Asn Asn Gly Pro 475 480 485 tgg gta act cac tgg gaa gaa atg gca aag aaa acg gct att cgt cgc 672 Trp Val Thr His Trp Glu Glu Met Ala Lys Lys Thr Ala Ile Arg Arg 490 495 500 505 ctg ttc aaa tat ttg ccc gta tca att gag atc cag cgt gca gta tca 720 Leu Phe Lys Tyr Leu Pro Val Ser Ile Glu Ile Gln Arg Ala Val Ser 510 515 520 atg gat gaa aag gaa cca ctg aca atc gat cct gca gat tcc tct gta 768 Met Asp Glu Lys Glu Pro Leu Thr Ile Asp Pro Ala Asp Ser Ser Val 525 530 535 tta acc ggg gaa tac agt gta atc gat aat tca gag gaa tag 810 Leu Thr Gly Glu Tyr Ser Val Ile Asp Asn Ser Glu Glu * 540 545 550 5 269 PRT Artificial Sequence misc_feature (1)..(269) recT on plasmid pBAD24-recET at 2155-2972 5 Met Thr Lys Gln Pro Pro Ile Ala Lys Ala Asp Leu Gln Lys Thr Gln 1 5 10 15 Gly Asn Arg Ala Pro Ala Ala Val Lys Asn Ser Asp Val Ile Ser Phe 20 25 30 Ile Asn Gln Pro Ser Met Lys Glu Gln Leu Ala Ala Ala Leu Pro Arg 35 40 45 His Met Thr Ala Glu Arg Met Ile Arg Ile Ala Thr Thr Glu Ile Arg 50 55 60 Lys Val Pro Ala Leu Gly Asn Cys Asp Thr Met Ser Phe Val Ser Ala 65 70 75 80 Ile Val Gln Cys Ser Gln Leu Gly Leu Glu Pro Gly Ser Ala Leu Gly 85 90 95 His Ala Tyr Leu Leu Pro Phe Gly Asn Lys Asn Glu Lys Ser Gly Lys 100 105 110 Lys Asn Val Gln Leu Ile Ile Gly Tyr Arg Gly Met Ile Asp Leu Ala 115 120 125 Arg Arg Ser Gly Gln Ile Ala Ser Leu Ser Ala Arg Val Val Arg Glu 130 135 140 Gly Asp Glu Phe Ser Phe Glu Phe Gly Leu Asp Glu Lys Leu Ile His 145 150 155 160 Arg Pro Gly Glu Asn Glu Asp Ala Pro Val Thr His Val Tyr Ala Val 165 170 175 Ala Arg Leu Lys Asp Gly Gly Thr Gln Phe Glu Val Met Thr Arg Lys 180 185 190 Gln Ile Glu Leu Val Arg Ser Leu Ser Lys Ala Gly Asn Asn Gly Pro 195 200 205 Trp Val Thr His Trp Glu Glu Met Ala Lys Lys Thr Ala Ile Arg Arg 210 215 220 Leu Phe Lys Tyr Leu Pro Val Ser Ile Glu Ile Gln Arg Ala Val Ser 225 230 235 240 Met Asp Glu Lys Glu Pro Leu Thr Ile Asp Pro Ala Asp Ser Ser Val 245 250 255 Leu Thr Gly Glu Tyr Ser Val Ile Asp Asn Ser Glu Glu 260 265 6 876 DNA Artificial Sequence misc_feature (1)..(876) araC on plasmid pBAD24-recET at 974-996 6 tgacaacttg acggctacat cattcacttt ttcttcacaa ccggcacgga actcgctcgg 60 gctggccccg gtgcattttt taaatacccg cgagaaatag agttgatcgt caaaaccaac 120 attgcgaccg acggtggcga taggcatccg ggtggtgctc aaaagcagct tcgcctggct 180 gatacgttgg tcctcgcgcc agcttaagac gctaatccct aactgctggc ggaaaagatg 240 tgacagacgc gacggcgaca agcaaacatg ctgtgcgacg ctggcgatat caaaattgct 300 gtctgccagg tgatcgctga tgtactgaca agcctcgcgt acccgattat ccatcggtgg 360 atggagcgac tcgttaatcg cttccatgcg ccgcagtaac aattgctcaa gcagatttat 420 cgccagcagc tccgaatagc gcccttcccc ttgcccggcg ttaatgattt gcccaaacag 480 gtcgctgaaa tgcggctggt gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat 540 tgacggccag ttaagccatt catgccagta ggcgcgcgga cgaaagtaaa cccactggtg 600 ataccattcg cgagcctccg gatgacgacc gtagtgatga atctctcctg gcgggaacag 660 caaaatatca cccggtcggc aaacaaattc tcgtccctga tttttcacca ccccctgacc 720 gcgaatggtg agattgagaa tataaccttt cattcccagc ggtcggtcga taaaaaaatc 780 gagataaccg ttggcctcaa tcggcgttaa acccgccacc agatgggcat taaacgagta 840 tcccggcagc aggggatcat tttgcgcttc agccat 876 7 292 PRT Artificial Sequence misc_feature (1)..(292) araC on plasmid pBAD24-recET at 974-996 7 Met Ala Glu Ala Gln Asn Asp Pro Leu Leu Pro Gly Tyr Ser Phe Asn 1 5 10 15 Ala His Leu Val Ala Gly Leu Thr Pro Ile Glu Ala Asn Gly Tyr Leu 20 25 30 Asp Phe Phe Ile Asp Arg Pro Leu Gly Met Lys Gly Tyr Ile Leu Asn 35 40 45 Leu Thr Ile Arg Gly Gln Gly Val Val Lys Asn Gln Gly Arg Glu Phe 50 55 60 Val Cys Arg Pro Gly Asp Ile Leu Leu Phe Pro Pro Gly Glu Ile His 65 70 75 80 His Tyr Gly Arg His Pro Glu Ala Arg Glu Trp Tyr His Gln Trp Val 85 90 95 Tyr Phe Arg Pro Arg Ala Tyr Trp His Glu Trp Leu Asn Trp Pro Ser 100 105 110 Ile Phe Ala Asn Thr Gly Phe Phe Arg Pro Asp Glu Ala His Gln Pro 115 120 125 His Phe Ser Asp Leu Phe Gly Gln Ile Ile Asn Ala Gly Gln Gly Glu 130 135 140 Gly Arg Tyr Ser Glu Leu Leu Ala Ile Asn Leu Leu Glu Gln Leu Leu 145 150 155 160 Leu Arg Arg Met Glu Ala Ile Asn Glu Ser Leu His Pro Pro Met Asp 165 170 175 Asn Arg Val Arg Glu Ala Cys Gln Tyr Ile Ser Asp His Leu Ala Asp 180 185 190 Ser Asn Phe Asp Ile Ala Ser Val Ala Gln His Val Cys Leu Ser Pro 195 200 205 Ser Arg Leu Ser His Leu Phe Arg Gln Gln Leu Gly Ile Ser Val Leu 210 215 220 Ser Trp Arg Glu Asp Gln Arg Ile Ser Gln Ala Lys Leu Leu Leu Ser 225 230 235 240 Thr Thr Arg Met Pro Ile Ala Thr Val Gly Arg Asn Val Gly Phe Asp 245 250 255 Asp Gln Leu Tyr Phe Ser Arg Val Phe Lys Lys Cys Thr Gly Ala Ser 260 265 270 Pro Ser Glu Phe Arg Ala Gly Cys Glu Glu Lys Val Asn Asp Val Ala 275 280 285 Val Lys Leu Ser 290 8 861 DNA Artificial Sequence misc_feature (1)..(861) bla gene on plasmid pBAD24-recET at 3493-4353 8 atg agt att caa cat ttc cgt gtc gcc ctt att ccc ttt ttt gcg gca 48 Met Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala 295 300 305 ttt tgc ctt cct gtt ttt gct cac cca gaa acg ctg gtg aaa gta aaa 96 Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys 310 315 320 gat gct gaa gat cag ttg ggt gca cga gtg ggt tac atc gaa ctg gat 144 Asp Ala Glu Asp Gln Leu Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp 325 330 335 340 ctc aac agc ggt aag atc ctt gag agt ttt cgc ccc gaa gaa cgt ttt 192 Leu Asn Ser Gly Lys Ile Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe 345 350 355 cca atg atg agc act ttt aaa gtt ctg cta tgt ggc gcg gta tta tcc 240 Pro Met Met Ser Thr Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser 360 365 370 cgt gtt gac gcc ggg caa gag caa ctc ggt cgc cgc ata cac tat tct 288 Arg Val Asp Ala Gly Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser 375 380 385 cag aat gac ttg gtt gag tac tca cca gtc aca gaa aag cat ctt acg 336 Gln Asn Asp Leu Val Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr 390 395 400 gat ggc atg aca gta aga gaa tta tgc agt gct gcc ata acc atg agt 384 Asp Gly Met Thr Val Arg Glu Leu Cys Ser Ala Ala Ile Thr Met Ser 405 410 415 420 gat aac act gcg gcc aac tta ctt ctg aca acg atc gga gga ccg aag 432 Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys 425 430 435 gag cta acc gct ttt ttg cac aac atg ggg gat cat gta act cgc ctt 480 Glu Leu Thr Ala Phe Leu His Asn Met Gly Asp His Val Thr Arg Leu 440 445 450 gat cgt tgg gaa ccg gag ctg aat gaa gcc ata cca aac gac gag cgt 528 Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg 455 460 465 gac acc acg atg cct gta gca atg gca aca acg ttg cgc aaa cta tta 576 Asp Thr Thr Met Pro Val Ala Met Ala Thr Thr Leu Arg Lys Leu Leu 470 475 480 act ggc gaa cta ctt act cta gct tcc cgg caa caa tta ata gac tgg 624 Thr Gly Glu Leu Leu Thr Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp 485 490 495 500 atg gag gcg gat aaa gtt gca gga cca ctt ctg cgc tcg gcc ctt ccg 672 Met Glu Ala Asp Lys Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro 505 510 515 gct ggc tgg ttt att gct gat aaa tct gga gcc ggt gag cgt ggg tct 720 Ala Gly Trp Phe Ile Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser 520 525 530 cgc ggt atc att gca gca ctg ggg cca gat ggt aag ccc tcc cgt atc 768 Arg Gly Ile Ile Ala Ala Leu Gly Pro Asp Gly Lys Pro Ser Arg Ile 535 540 545 gta gtt atc tac acg acg ggg agt cag gca act atg gat gaa cga aat 816 Val Val Ile Tyr Thr Thr Gly Ser Gln Ala Thr Met Asp Glu Arg Asn 550 555 560 aga cag atc gct gag ata ggt gcc tca ctg att aag cat tgg taa 861 Arg Gln Ile Ala Glu Ile Gly Ala Ser Leu Ile Lys His Trp * 565 570 575 9 286 PRT Artificial Sequence DOMAIN (1)..(286) bla gene on plasmid pBAD24-recET at 3493-4353 9 Met Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala 1 5 10 15 Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys 20 25 30 Asp Ala Glu Asp Gln Leu Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp 35 40 45 Leu Asn Ser Gly Lys Ile Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe 50 55 60 Pro Met Met Ser Thr Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser 65 70 75 80 Arg Val Asp Ala Gly Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser 85 90 95 Gln Asn Asp Leu Val Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr 100 105 110 Asp Gly Met Thr Val Arg Glu Leu Cys Ser Ala Ala Ile Thr Met Ser 115 120 125 Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys 130 135 140 Glu Leu Thr Ala Phe Leu His Asn Met Gly Asp His Val Thr Arg Leu 145 150 155 160 Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg 165 170 175 Asp Thr Thr Met Pro Val Ala Met Ala Thr Thr Leu Arg Lys Leu Leu 180 185 190 Thr Gly Glu Leu Leu Thr Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp 195 200 205 Met Glu Ala Asp Lys Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro 210 215 220 Ala Gly Trp Phe Ile Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser 225 230 235 240 Arg Gly Ile Ile Ala Ala Leu Gly Pro Asp Gly Lys Pro Ser Arg Ile 245 250 255 Val Val Ile Tyr Thr Thr Gly Ser Gln Ala Thr Met Asp Glu Arg Asn 260 265 270 Arg Gln Ile Ala Glu Ile Gly Ala Ser Leu Ile Lys His Trp 275 280 285 10 7195 DNA Artificial Sequence misc_feature (1)..(7195) plasmid pBAD-ET-gamma 10 atcgatgcat aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac 60 tccgtcaagc cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca 120 ttcacttttt cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta 180 aatacccgcg agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata 240 ggcatccggg tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag 300 cttaagacgc taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag 360 caaacatgct gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg 420 tactgacaag cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct 480 tccatgcgcc gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc 540 ccttcccctt gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc 600 gcttcatccg ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca 660 tgccagtagg cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga 720 tgacgaccgt agtgatgaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa 780 acaaattctc gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata 840 taacctttca ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc 900 ggcgttaaac ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt 960 tgcgcttcag ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat 1020 tgcatcagac attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta 1080 accccgctta ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt 1140 aacaaaagtg tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca 1200 ctttgctatg ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta 1260 tcgcaactct ctactgtttc tccatacccg tttttttggg ctagcaggag gaattcacca 1320 tggatcccgt aatcgtagaa gacatagagc caggtattta ttacggaatt tcgaatgaga 1380 attaccacgc gggtcccggt atcagtaagt ctcagctcga tgacattgct gatactccgg 1440 cactatattt gtggcgtaaa aatgcccccg tggacaccac aaagacaaaa acgctcgatt 1500 taggaactgc tttccactgc cgggtacttg aaccggaaga attcagtaac cgctttatcg 1560 tagcacctga atttaaccgc cgtacaaacg ccggaaaaga agaagagaaa gcgtttctga 1620 tggaatgcgc aagcacagga aaaacggtta tcactgcgga agaaggccgg aaaattgaac 1680 tcatgtatca aagcgttatg gctttgccgc tggggcaatg gcttgttgaa agcgccggac 1740 acgctgaatc atcaatttac tgggaagatc ctgaaacagg aattttgtgt cggtgccgtc 1800 cggacaaaat tatccctgaa tttcactgga tcatggacgt gaaaactacg gcggatattc 1860 aacgattcaa aaccgcttat tacgactacc gctatcacgt tcaggatgca ttctacagtg 1920 acggttatga agcacagttt ggagtgcagc caactttcgt ttttctggtt gccagcacaa 1980 ctattgaatg cggacgttat ccggttgaaa ttttcatgat gggcgaagaa gcaaaactgg 2040 caggtcaaca ggaatatcac cgcaatctgc gaaccctgtc tgactgcctg aataccgatg 2100 aatggccagc tattaagaca ttatcactgc cccgctgggc taaggaatat gcaaatgact 2160 agatctcgag gtacccgagc acgtgttgac aattaatcat cggcatagta tatcggcata 2220 gtataatacg acaaggtgag gaactaaacc atggctaagc aaccaccaat cgcaaaagcc 2280 gatctgcaaa aaactcaggg aaaccgtgca ccagcagcag ttaaaaatag cgacgtgatt 2340 agttttatta accagccatc aatgaaagag caactggcag cagctcttcc acgccatatg 2400 acggctgaac gtatgatccg tatcgccacc acagaaattc gtaaagttcc ggcgttagga 2460 aactgtgaca ctatgagttt tgtcagtgcg atcgtacagt gttcacagct cggacttgag 2520 ccaggtagcg ccctcggtca tgcatattta ctgccttttg gtaataaaaa cgaaaagagc 2580 ggtaaaaaga acgttcagct aatcattggc tatcgcggca tgattgatct ggctcgccgt 2640 tctggtcaaa tcgccagcct gtcagcccgt gttgtccgtg aaggtgacga gtttagcttc 2700 gaatttggcc ttgatgaaaa gttaatacac cgcccgggag aaaacgaaga tgccccggtt 2760 acccacgtct atgctgtcgc aagactgaaa gacggaggta ctcagtttga agttatgacg 2820 cgcaaacaga ttgagctggt gcgcagcctg agtaaagctg gtaataacgg gccgtgggta 2880 actcactggg aagaaatggc aaagaaaacg gctattcgtc gcctgttcaa atatttgccc 2940 gtatcaattg agatccagcg tgcagtatca atggatgaaa aggaaccact gacaatcgat 3000 cctgcagatt cctctgtatt aaccggggaa tacagtgtaa tcgataattc agaggaatag 3060 atctaagctt cctgctgaac atcaaaggca agaaaacatc tgttgtcaaa gacagcatcc 3120 ttgaacaagg acaattaaca gttaacaaat aaaaacgcaa aagaaaatgc cgatatccta 3180 ttggcatttt cttttatttc ttatcaacat aaaggtgaat cccatacctc gagcttcacg 3240 ctgccgcaag cactcagggc gcaagggctg ctaaaaggaa gcggaacacg tagaaagcca 3300 gtccgcagaa acggtgctga ccccggatga atgtcagcta ctgggctatc tggacaaggg 3360 aaaacgcaag cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg cgatagctag 3420 actgggcggt tttatggaca gcaagcgaac cggaattgcc agctggggcg ccctctggta 3480 aggttgggaa gccctgcaaa gtaaactgga tggctttctt gccgccaagg atctgatggc 3540 gcaggggatc aagatctgat caagagacag gatgaggatc gtttcgcatg gatattaata 3600 ctgaaactga gatcaagcaa aagcattcac taaccccctt tcctgttttc ctaatcagcc 3660 cggcatttcg cgggcgatat tttcacagct atttcaggag ttcagccatg aacgcttatt 3720 acattcagga tcgtcttgag gctcagagct gggcgcgtca ctaccagcag ctcgcccgtg 3780 aagagaaaga ggcagaactg gcagacgaca tggaaaaagg cctgccccag cacctgtttg 3840 aatcgctatg catcgatcat ttgcaacgcc acggggccag caaaaaatcc attacccgtg 3900 cgtttgatga cgatgttgag tttcaggagc gcatggcaga acacatccgg tacatggttg 3960 aaaccattgc tcaccaccag gttgatattg attcagaggt ataaaacgag tagaagcttg 4020 gctgttttgg cggatgagag aagattttca gcctgataca gattaaatca gaacgcagaa 4080 gcggtctgat aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca cctgacccca 4140 tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag tgtggggtct ccccatgcga 4200 gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt 4260 cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg 4320 gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact 4380 gccaggcatc aaattaagca gaaggccatc ctgacggatg gcctttttgc gtttctacaa 4440 actcttttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc 4500 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt 4560 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct 4620 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga 4680 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag 4740 cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca 4800 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga 4860 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag 4920 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc 4980 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa 5040 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt 5100 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg 5160 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt 5220 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg 5280 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat 5340 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact 5400 gtcagaccaa gtttactcat atatacttta gattgattta cgcgccctgt agcggcgcat 5460 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 5520 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 5580 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 5640 ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt 5700 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaacttgaa 5760 caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg 5820 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 5880 taacgtttac aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 5940 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 6000 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 6060 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 6120 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 6180 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 6240 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 6300 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 6360 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 6420 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 6480 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 6540 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 6600 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 6660 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 6720 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc 6780 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat agggtcatgg 6840 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg 6900 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac 6960 cgtcatcacc gaaacgcgcg aggcagcaag gagatggcgc ccaacagtcc cccggccacg 7020 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga 7080 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg 7140 atgccggcca cgatgcgtcc ggcgtagagg atctgctcat gtttgacagc ttatc 7195 11 7010 DNA Artificial Sequence misc_feature (1)..(7010) plasmid pBAD-alpha-beta-gamma 11 atcgatgcat aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac 60 tccgtcaagc cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca 120 ttcacttttt cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta 180 aatacccgcg agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata 240 ggcatccggg tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag 300 cttaagacgc taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag 360 caaacatgct gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg 420 tactgacaag cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct 480 tccatgcgcc gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc 540 ccttcccctt gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc 600 gcttcatccg ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca 660 tgccagtagg cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga 720 tgacgaccgt agtgatgaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa 780 acaaattctc gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata 840 taacctttca ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc 900 ggcgttaaac ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt 960 tgcgcttcag ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat 1020 tgcatcagac attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta 1080 accccgctta ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt 1140 aacaaaagtg tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca 1200 ctttgctatg ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta 1260 tcgcaactct ctactgtttc tccatacccg tttttttggg ctagcaggag gaattcacc 1319 atg aca ccg gac att atc ctg cag cgt acc ggg atc gat gtg aga gct 1367 Met Thr Pro Asp Ile Ile Leu Gln Arg Thr Gly Ile Asp Val Arg Ala 290 295 300 gtc gaa cag ggg gat gat gcg tgg cac aaa tta cgg ctc ggc gtc atc 1415 Val Glu Gln Gly Asp Asp Ala Trp His Lys Leu Arg Leu Gly Val Ile 305 310 315 acc gct tca gaa gtt cac aac gtg ata gca aaa ccc cgc tcc gga aag 1463 Thr Ala Ser Glu Val His Asn Val Ile Ala Lys Pro Arg Ser Gly Lys 320 325 330 335 aag tgg cct gac atg aaa atg tcc tac ttc cac acc ctg ctt gct gag 1511 Lys Trp Pro Asp Met Lys Met Ser Tyr Phe His Thr Leu Leu Ala Glu 340 345 350 gtt tgc acc ggt gtg gct ccg gaa gtt aac gct aaa gca ctg gcc tgg 1559 Val Cys Thr Gly Val Ala Pro Glu Val Asn Ala Lys Ala Leu Ala Trp 355 360 365 gga aaa cag tac gag aac gac gcc aga acc ctg ttt gaa ttc act tcc 1607 Gly Lys Gln Tyr Glu Asn Asp Ala Arg Thr Leu Phe Glu Phe Thr Ser 370 375 380 ggc gtg aat gtt act gaa tcc ccg atc atc tat cgc gac gaa agt atg 1655 Gly Val Asn Val Thr Glu Ser Pro Ile Ile Tyr Arg Asp Glu Ser Met 385 390 395 cgt acc gcc tgc tct ccc gat ggt tta tgc agt gac ggc aac ggc ctt 1703 Arg Thr Ala Cys Ser Pro Asp Gly Leu Cys Ser Asp Gly Asn Gly Leu 400 405 410 415 gaa ctg aaa tgc ccg ttt acc tcc cgg gat ttc atg aag ttc cgg ctc 1751 Glu Leu Lys Cys Pro Phe Thr Ser Arg Asp Phe Met Lys Phe Arg Leu 420 425 430 ggt ggt ttc gag gcc ata aag tca gct tac atg gcc cag gtg cag tac 1799 Gly Gly Phe Glu Ala Ile Lys Ser Ala Tyr Met Ala Gln Val Gln Tyr 435 440 445 agc atg tgg gtg acg cga aaa aat gcc tgg tac ttt gcc aac tat gac 1847 Ser Met Trp Val Thr Arg Lys Asn Ala Trp Tyr Phe Ala Asn Tyr Asp 450 455 460 ccg cgt atg aag cgt gaa ggc ctg cat tat gtc gtg att gag cgg gat 1895 Pro Arg Met Lys Arg Glu Gly Leu His Tyr Val Val Ile Glu Arg Asp 465 470 475 gaa aag tac atg gcg agt ttt gac gag atc gtg ccg gag ttc atc gaa 1943 Glu Lys Tyr Met Ala Ser Phe Asp Glu Ile Val Pro Glu Phe Ile Glu 480 485 490 495 aaa atg gac gag gca ctg gct gaa att ggt ttt gta ttt ggg gag caa 1991 Lys Met Asp Glu Ala Leu Ala Glu Ile Gly Phe Val Phe Gly Glu Gln 500 505 510 tgg cga tag atccggtacc cgagcacgtg ttgacaatta atcatcggca 2040 Trp Arg * tagtatatcg gcatagtata atacgacaag gtgaggaact aaacc atg agt act 2094 Met Ser Thr 1 gca ctc gca acg ctg gct ggg aag ctg gct gaa cgt gtc ggc atg gat 2142 Ala Leu Ala Thr Leu Ala Gly Lys Leu Ala Glu Arg Val Gly Met Asp 5 10 15 tct gtc gac cca cag gaa ctg atc acc act ctt cgc cag acg gca ttt 2190 Ser Val Asp Pro Gln Glu Leu Ile Thr Thr Leu Arg Gln Thr Ala Phe 20 25 30 35 aaa ggt gat gcc agc gat gcg cag ttc atc gca tta ctg atc gtt gcc 2238 Lys Gly Asp Ala Ser Asp Ala Gln Phe Ile Ala Leu Leu Ile Val Ala 40 45 50 aac cag tac ggc ctt aat ccg tgg acg aaa gaa att tac gcc ttt cct 2286 Asn Gln Tyr Gly Leu Asn Pro Trp Thr Lys Glu Ile Tyr Ala Phe Pro 55 60 65 gat aag cag aat ggc atc gtt ccg gtg gtg ggc gtt gat ggc tgg tcc 2334 Asp Lys Gln Asn Gly Ile Val Pro Val Val Gly Val Asp Gly Trp Ser 70 75 80 cgc atc atc aat gaa aac cag cag ttt gat ggc atg gac ttt gag cag 2382 Arg Ile Ile Asn Glu Asn Gln Gln Phe Asp Gly Met Asp Phe Glu Gln 85 90 95 gac aat gaa tcc tgt aca tgc cgg att tac cgc aag gac cgt aat cat 2430 Asp Asn Glu Ser Cys Thr Cys Arg Ile Tyr Arg Lys Asp Arg Asn His 100 105 110 115 ccg atc tgc gtt acc gaa tgg atg gat gaa tgc cgc cgc gaa cca ttc 2478 Pro Ile Cys Val Thr Glu Trp Met Asp Glu Cys Arg Arg Glu Pro Phe 120 125 130 aaa act cgc gaa ggc aga gaa atc acg ggg ccg tgg cag tcg cat ccc 2526 Lys Thr Arg Glu Gly Arg Glu Ile Thr Gly Pro Trp Gln Ser His Pro 135 140 145 aaa cgg atg tta cgt cat aaa gcc atg att cag tgt gcc cgt ctg gcc 2574 Lys Arg Met Leu Arg His Lys Ala Met Ile Gln Cys Ala Arg Leu Ala 150 155 160 ttc gga ttt gct ggt atc tat gac aag gat gaa gcc gag cgc att gtc 2622 Phe Gly Phe Ala Gly Ile Tyr Asp Lys Asp Glu Ala Glu Arg Ile Val 165 170 175 gaa aat act gca tac act gca gaa cgt cag ccg gaa cgc gac atc act 2670 Glu Asn Thr Ala Tyr Thr Ala Glu Arg Gln Pro Glu Arg Asp Ile Thr 180 185 190 195 ccg gtt aac gat gaa acc atg cag gag att aac act ctg ctg atc gcc 2718 Pro Val Asn Asp Glu Thr Met Gln Glu Ile Asn Thr Leu Leu Ile Ala 200 205 210 ctg gat aaa aca tgg gat gac gac tta ttg ccg ctc tgt tcc cag ata 2766 Leu Asp Lys Thr Trp Asp Asp Asp Leu Leu Pro Leu Cys Ser Gln Ile 215 220 225 ttt cgc cgc gac att cgt gca tcg tca gaa ctg aca cag gcc gaa gca 2814 Phe Arg Arg Asp Ile Arg Ala Ser Ser Glu Leu Thr Gln Ala Glu Ala 230 235 240 gta aaa gct ctt gga ttc ctg aaa cag aaa gcc gca gag cag aag gtg 2862 Val Lys Ala Leu Gly Phe Leu Lys Gln Lys Ala Ala Glu Gln Lys Val 245 250 255 gca gca tag atctcgagaa gcttcctgct gaacatcaaa ggcaagaaaa 2911 Ala Ala * 260 catctgttgt caaagacagc atccttgaac aaggacaatt aacagttaac aaataaaaac 2971 gcaaaagaaa atgccgatat cctattggca ttttctttta tttcttatca acataaaggt 3031 gaatcccata cctcgagctt cacgctgccg caagcactca gggcgcaagg gctgctaaaa 3091 ggaagcggaa cacgtagaaa gccagtccgc agaaacggtg ctgaccccgg atgaatgtca 3151 gctactgggc tatctggaca agggaaaacg caagcgcaaa gagaaagcag gtagcttgca 3211 gtgggcttac atggcgatag ctagactggg cggttttatg gacagcaagc gaaccggaat 3271 tgccagctgg ggcgccctct ggtaaggttg ggaagccctg caaagtaaac tggatggctt 3331 tcttgccgcc aaggatctga tggcgcaggg gatcaagatc tgatcaagag acaggatgag 3391 gatcgtttcg c atg gat att aat act gaa act gag atc aag caa aag cat 3441 Met Asp Ile Asn Thr Glu Thr Glu Ile Lys Gln Lys His 1 5 10 tca cta acc ccc ttt cct gtt ttc cta atc agc ccg gca ttt cgc ggg 3489 Ser Leu Thr Pro Phe Pro Val Phe Leu Ile Ser Pro Ala Phe Arg Gly 15 20 25 cga tat ttt cac agc tat ttc agg agt tca gcc atg aac gct tat tac 3537 Arg Tyr Phe His Ser Tyr Phe Arg Ser Ser Ala Met Asn Ala Tyr Tyr 30 35 40 45 att cag gat cgt ctt gag gct cag agc tgg gcg cgt cac tac cag cag 3585 Ile Gln Asp Arg Leu Glu Ala Gln Ser Trp Ala Arg His Tyr Gln Gln 50 55 60 ctc gcc cgt gaa gag aaa gag gca gaa ctg gca gac gac atg gaa aaa 3633 Leu Ala Arg Glu Glu Lys Glu Ala Glu Leu Ala Asp Asp Met Glu Lys 65 70 75 ggc ctg ccc cag cac ctg ttt gaa tcg cta tgc atc gat cat ttg caa 3681 Gly Leu Pro Gln His Leu Phe Glu Ser Leu Cys Ile Asp His Leu Gln 80 85 90 cgc cac ggg gcc agc aaa aaa tcc att acc cgt gcg ttt gat gac gat 3729 Arg His Gly Ala Ser Lys Lys Ser Ile Thr Arg Ala Phe Asp Asp Asp 95 100 105 gtt gag ttt cag gag cgc atg gca gaa cac atc cgg tac atg gtt gaa 3777 Val Glu Phe Gln Glu Arg Met Ala Glu His Ile Arg Tyr Met Val Glu 110 115 120 125 acc att gct cac cac cag gtt gat att gat tca gag gta taa 3819 Thr Ile Ala His His Gln Val Asp Ile Asp Ser Glu Val * 130 135 aacgagtaga agcttggctg ttttggcgga tgagagaaga ttttcagcct gatacagatt 3879 aaatcagaac gcagaagcgg tctgataaaa cagaatttgc ctggcggcag tagcgcggtg 3939 gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg 3999 gggtctcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc 4059 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac 4119 aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg 4179 acgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct 4239 ttttgcgttt ctacaaactc ttttgtttat ttttctaaat acattcaaat atgtatccgc 4299 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 4359 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 4419 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 4479 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 4539 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg 4599 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 4659 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 4719 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 4779 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 4839 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag 4899 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 4959 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 5019 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 5079 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 5139 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 5199 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttacgcg 5259 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca 5319 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc 5379 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct 5439 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg 5499 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc 5559 ttgttccaaa cttgaacaac actcaaccct atctcgggct attcttttga tttataaggg 5619 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg 5679 aattttaaca aaatattaac gtttacaatt taaaaggatc taggtgaaga tcctttttga 5739 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 5799 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 5859 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 5919 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta 5979 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 6039 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 6099 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 6159 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 6219 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 6279 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 6339 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 6399 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 6459 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 6519 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 6579 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca 6639 ccgcataggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg 6699 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 6759 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcaaggaga tggcgcccaa 6819 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc 6879 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc 6939 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatct gctcatgttt 6999 gacagcttat c 7010 12 226 PRT Artificial Sequence DOMAIN (1)..(226) Red-alpha from plasmid pBAD-alpha-beta-gamma 12 Met Thr Pro Asp Ile Ile Leu Gln Arg Thr Gly Ile Asp Val Arg Ala 1 5 10 15 Val Glu Gln Gly Asp Asp Ala Trp His Lys Leu Arg Leu Gly Val Ile 20 25 30 Thr Ala Ser Glu Val His Asn Val Ile Ala Lys Pro Arg Ser Gly Lys 35 40 45 Lys Trp Pro Asp Met Lys Met Ser Tyr Phe His Thr Leu Leu Ala Glu 50 55 60 Val Cys Thr Gly Val Ala Pro Glu Val Asn Ala Lys Ala Leu Ala Trp 65 70 75 80 Gly Lys Gln Tyr Glu Asn Asp Ala Arg Thr Leu Phe Glu Phe Thr Ser 85 90 95 Gly Val Asn Val Thr Glu Ser Pro Ile Ile Tyr Arg Asp Glu Ser Met 100 105 110 Arg Thr Ala Cys Ser Pro Asp Gly Leu Cys Ser Asp Gly Asn Gly Leu 115 120 125 Glu Leu Lys Cys Pro Phe Thr Ser Arg Asp Phe Met Lys Phe Arg Leu 130 135 140 Gly Gly Phe Glu Ala Ile Lys Ser Ala Tyr Met Ala Gln Val Gln Tyr 145 150 155 160 Ser Met Trp Val Thr Arg Lys Asn Ala Trp Tyr Phe Ala Asn Tyr Asp 165 170 175 Pro Arg Met Lys Arg Glu Gly Leu His Tyr Val Val Ile Glu Arg Asp 180 185 190 Glu Lys Tyr Met Ala Ser Phe Asp Glu Ile Val Pro Glu Phe Ile Glu 195 200 205 Lys Met Asp Glu Ala Leu Ala Glu Ile Gly Phe Val Phe Gly Glu Gln 210 215 220 Trp Arg 225 13 261 PRT Artificial Sequence DOMAIN (1)..(261) Red-beta from plasmid pBAD-alpha-beta-gamma 13 Met Ser Thr Ala Leu Ala Thr Leu Ala Gly Lys Leu Ala Glu Arg Val 1 5 10 15 Gly Met Asp Ser Val Asp Pro Gln Glu Leu Ile Thr Thr Leu Arg Gln 20 25 30 Thr Ala Phe Lys Gly Asp Ala Ser Asp Ala Gln Phe Ile Ala Leu Leu 35 40 45 Ile Val Ala Asn Gln Tyr Gly Leu Asn Pro Trp Thr Lys Glu Ile Tyr 50 55 60 Ala Phe Pro Asp Lys Gln Asn Gly Ile Val Pro Val Val Gly Val Asp 65 70 75 80 Gly Trp Ser Arg Ile Ile Asn Glu Asn Gln Gln Phe Asp Gly Met Asp 85 90 95 Phe Glu Gln Asp Asn Glu Ser Cys Thr Cys Arg Ile Tyr Arg Lys Asp 100 105 110 Arg Asn His Pro Ile Cys Val Thr Glu Trp Met Asp Glu Cys Arg Arg 115 120 125 Glu Pro Phe Lys Thr Arg Glu Gly Arg Glu Ile Thr Gly Pro Trp Gln 130 135 140 Ser His Pro Lys Arg Met Leu Arg His Lys Ala Met Ile Gln Cys Ala 145 150 155 160 Arg Leu Ala Phe Gly Phe Ala Gly Ile Tyr Asp Lys Asp Glu Ala Glu 165 170 175 Arg Ile Val Glu Asn Thr Ala Tyr Thr Ala Glu Arg Gln Pro Glu Arg 180 185 190 Asp Ile Thr Pro Val Asn Asp Glu Thr Met Gln Glu Ile Asn Thr Leu 195 200 205 Leu Ile Ala Leu Asp Lys Thr Trp Asp Asp Asp Leu Leu Pro Leu Cys 210 215 220 Ser Gln Ile Phe Arg Arg Asp Ile Arg Ala Ser Ser Glu Leu Thr Gln 225 230 235 240 Ala Glu Ala Val Lys Ala Leu Gly Phe Leu Lys Gln Lys Ala Ala Glu 245 250 255 Gln Lys Val Ala Ala 260 14 138 PRT Artificial Sequence DOMAIN (1)..(138) Red-gamma from plasmid pBAD-alpha-beta-gamma and plasmid pBAD-ET-gamma 14 Met Asp Ile Asn Thr Glu Thr Glu Ile Lys Gln Lys His Ser Leu Thr 1 5 10 15 Pro Phe Pro Val Phe Leu Ile Ser Pro Ala Phe Arg Gly Arg Tyr Phe 20 25 30 His Ser Tyr Phe Arg Ser Ser Ala Met Asn Ala Tyr Tyr Ile Gln Asp 35 40 45 Arg Leu Glu Ala Gln Ser Trp Ala Arg His Tyr Gln Gln Leu Ala Arg 50 55 60 Glu Glu Lys Glu Ala Glu Leu Ala Asp Asp Met Glu Lys Gly Leu Pro 65 70 75 80 Gln His Leu Phe Glu Ser Leu Cys Ile Asp His Leu Gln Arg His Gly 85 90 95 Ala Ser Lys Lys Ser Ile Thr Arg Ala Phe Asp Asp Asp Val Glu Phe 100 105 110 Gln Glu Arg Met Ala Glu His Ile Arg Tyr Met Val Glu Thr Ile Ala 115 120 125 His His Gln Val Asp Ile Asp Ser Glu Val 130 135 

1. A method for cloning DNA molecules in cells comprising the steps of: a) providing a host cell capable of performing homologous recombination, b) contacting in said host cell a first DNA molecule which is capable of being replicated in said host cell with a second DNA molecule comprising at least two regions of sequence homology to regions on the first DNA molecule, under conditions which favour homologous recombination between said first and second DNA molecules and c) selecting a host cell in which homologous recombination between said first and second DNA molecules has occurred.
 2. The method according to claim 1 wherein the homologous recombination occurs via the recET cloning mechanism.
 3. The method according to claim 2 wherein the host cell is capable of expressing recE and recT genes.
 4. The method according to claim 3 wherein the recE and recT genes are selected from E. coli recE and recT genes or from λ redα and redβ genes.
 5. The method according to claim 3 or 4 wherein the host cell is transformed with at least one vector capable of expressing recE and/or recT genes.
 6. The method of claim 3, 4 or 5 wherein the expression of the recE and/or recT genes is under control of a regulatable promoter.
 7. The method of claim 5 or 6 wherein the recT gene is overexpressed versus the recE gene.
 8. The method according to any one of claims 3 to 7 wherein the recE gene is selected from a nucleic acid molecule comprising (a) the nucleic acid sequence from position 1320 (ATG) to 2159 (GAC) as depicted in FIG. 7B, (b) the nucleic acid sequence from position 1320 (ATG) to 1998 (CGA) as depicted in FIG. 13B, (c) a nucleic acid encoding the same polypeptide within the degeneracy of the genetic code and/or (d) a nucleic acid sequence which hybridizes under stringent conditions with the nucleic acid sequence from (a), (b) and/or (c).
 9. The method according to any one of claims 3 to 8 wherein the recT gene is selected from a nucleic acid molecule comprising (a) the nucleic acid sequence from position 2155 (ATG) to 2961 (GAA) as depicted in FIG. 7B, (b) the nucleic acid sequence from position 2086 (ATG) to 2868 (GCA) as depicted in FIG. 13B, (c) a nucleic acid encoding the same polypeptide within the degeneracy of the genetic code and/or (d) a nucleic acid sequence which hybridizes under stringent conditions with the nucleic acid sequences from (a), (b) and/or (c).
 10. The method according to any one of the previous claims wherein the host cell is a gram-negative bacterial cell.
 11. The method according to claim 10 wherein the host cell is an Escherichia coli cell.
 12. The method according to claim 11 wherein the host cell is an Escherichia coli K12 strain.
 13. The method according to claim 12 wherein the E. coli strain is s selected from JC 8679 and JC
 9604. 14. The method according to any one of the previous claims wherein the host cell further is capable of expressing a recBC inhibitor gene.
 15. The method according to claim 14 wherein the host cell is transformed with a vector expressing the recBC inhibitor gene.
 16. The method according to claim 14 or 15 wherein the recBC inhibitor gene is selected from a nucleic acid molecule comprising (a) the nucleic acid sequence from position 3588 (ATG) to 4002 (GTA) as depicted in FIG. 13B, (b) a nucleic acid encoding the same polypeptide within the degeneracy of the genetic code and/or (c) a nucleic acid sequence which hybridizes under stringent conditions (as defined above) with the nucleic acid sequence from (a) and/or (b).
 17. The method according to any one of claims 13 to 16 wherein the host cell is a prokaryotic recBC+ cell.
 18. The method according to any one of the previous claims wherein the first DNA molecule is circular.
 19. The method according to any one of the previous claims wherein the first DNA molecule is an extrachromosomal DNA molecule containing an origin of replication which is operative in the host cell.
 20. The method according to claim 18 or 19 wherein the first DNA molecule is selected from plasmids, cosmids, P1 vectors, BAC vectors and PAC vectors.
 21. The method according to any one of claims 1-18 wherein the first DNA molecule is a host cell chromosome.
 22. The method according to any one of the previous claims wherein the second DNA molecule is linear.
 23. The method according to any one of the previous claims wherein the regions of sequence homology are at least 15 nucleotides each.
 24. The method according to one of claims 1 to 16 wherein the second DNA molecule is obtained by an amplification reaction.
 25. The method according to one of the previous claims wherein the first and/or second DNA molecules are introduced into the host cells by transformation.
 26. The method according to claim 25 wherein the transformation method is electroporation.
 27. The method according to one of claims 1 to 26 wherein the first and second DNA molecules are introduced into the host cell simultaneously by co-transformation.
 28. The method according to one of claims 1 to 26 wherein the second DNA molecule is introduced into a host cell in which the first DNA molecule is already present.
 29. The method according to one of the previous claims wherein the second DNA molecule contains at least one marker gene placed between the two regions of sequence homology and wherein homologous recombination is detected by expression of said marker gene.
 30. The method according to claim 29 wherein gene presence is selected from antibiotic resistance genes, deficiency complementation genes and reporter genes.
 31. The method of any one of claims 1 to 30 wherein the first DNA molecule contains at least one marker gene between the two regions of sequence homology and wherein homologous recombination is detected by lack of expression of said marker gene.
 32. The method of any one of claims 1 to 31 wherein said marker gene is selected from genes which, under selected conditions, convey a toxic or bacteriostatic effect on the cell, and reporter genes.
 33. A method according to any one of the previous claims wherein the first DNA molecule contains at least one target site for a site specific recombinase between the two regions of sequence homology and wherein homologous recombination is detected by removal of said target site.
 34. A method for cloning DNA molecules comprising the steps of: (a) providing a source of RecE and RecT proteins, (b) contacting a first DNA molecule which is capable of being replicated in a suitable host cell with a second DNA molecule comprising at least two regions of sequence homology to regions on the first DNA molecule, under conditions which favour homologous recombination between said first and second DMA molecules and (c) selecting DNA molecules in which homologous recombination between said first and second DNA molecules has occurred.
 35. The method of claim 34 wherein said RecE and RecT or proteins are s selected from E. coli RecE and RecT proteins or from phage λ Redα and Redβ proteins.
 36. The method of claim 34 or 35 wherein the recombination occurs in vitro.
 37. The method of claim 34 or 35 wherein the recombination occurs in vivo.
 38. Use of cells capable of expressing the recE and recT genes as a host cell for a cloning method involving homologous recombination.
 39. Use of a vector system capable of expressing recE and recT genes in a host cell for a cloning method involving homologous recombination.
 40. Use of claims 38 or 39 wherein the recE and recT genes are selected from E. coli recE and recT genes or from λ redα and redβ genes.
 41. Use of a source of RecE and RecT proteins for a cloning method involving homologous recombination.
 42. Use of claim 41 wherein said RecE and RecT or proteins are selected from E. coli RecE and RecT proteins or from phage λ Redα and Redβ proteins.
 43. A reagent kit for cloning comprising (a) a host cell (b) means of expressing recE and recT genes in said host cell and (c) a recipient cloning vehicle capable of being replicated in said cell.
 44. The reagent kit according to claim 43 wherein the means (b) comprise a vector system capable of expressing the recE and recT genes in the host cell.
 45. The reagent kit according to claim 43 or 44 wherein the recE and recT genes are selected from E. coli recE and recT genes or from λ redα and redβ genes.
 46. A reagent kit for cloning comprising (a) a source for RecE and RecT proteins and (b) a recipient cloning vehicle capable of being propagated in a host cell.
 47. The reagent kit according to claim 46 further comprising a host cell suitable for propagating said recipient cloning vehicle.
 48. The reagent kit according to claim 46 or 47 wherein said RecE and RecT or proteins are selected from E. coli RecE and RecT proteins or from phage λ Redα and Redβ proteins.
 49. The reagent kit according to any one of claims 43-48 further comprising means for expressing a site specific recombinase in said host cell.
 50. The reagent kit according to any one of claims 43-49 further comprising nucleic acid amplification primers comprising a region of homology to said recipient cloning vehicle. 